Method and Compositions for Improved Lignocellulosic Material Hydrolysis

ABSTRACT

A method of digesting a lignocellulosic material is disclosed. In one embodiment, the method comprises the step of exposing the material to an effective amount of  Streptomyces  sp. ActE secretome such that at least partial lignocellulosic digestion occurs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit from U.S. Provisional Application61/579,301 filed Dec. 22, 2011 and U.S. Provisional Application61/579,897 filed Dec. 23, 2011, both of which are incorporated herein byreference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under DE-FC02-07ER64494awarded by the US Department of Energy and GM094584 awarded by theNational Institutes of Health. The government has certain rights in theinvention.

BACKGROUND

Cellulose is the most abundant organic polymer on Earth and represents avast source of renewable energy. Most of this energy is stored in therecalcitrant polysaccharide cellulose, which is difficult to hydrolyzebecause of the highly crystalline structure, and in hemicellulose, whichpresents challenges because of its structural diversity and complexity.Plant cell walls are approximately composed in pinewood of lignin (30%by weight), hemicellulose (glucomannan, 20%, arabinoxylan, 10%), andcrystalline cellulose (40%), which presents a major barrier to efficientuse. In terrestrial ecosystems, cellulolytic microbes help drive carboncycling through the deconstruction of biomass into simple sugars. Thedeconstruction is largely accomplished through the action ofcombinations of secreted glycoside hydrolases (GHs), carbohydrateesterases (CEs), polysaccharide lyases (PLs), and carbohydrate bindingmodules (CBMs) (Baldrian and Valaskova, 2008; Cantarel, et al., 2009;Lynd, Weimer, et al., 2002; Schuster and Schmoll, 2010). Consequently,organisms from many lignocellulose-rich environments and their enzymesare being studied for new insights into overcoming this barrier.

In order to obtain the hydrolysis of crystalline cellulose, enzymes mustcleave three types of glycosidic bonds. These enzymes areendocellulases, which cleave beta-1,4 glycosidic bonds that residewithin intact cellulose strands in the crystalline face,non-reducing-end exocellulases, which remove cellobiose units from thenon-reducing end of cellulose strands, and reducing-end exocellulases,which remove glycosyl units from the reducing-end of a cellulose strand.The endocellulolytic reaction is essential because it creates thenon-reducing and reducing ends that serve as the starting point forexocellulolytic reactions. The exocellulolytic reactions are essentialbecause they remove glycosyl groups in a processive manner from thebreakages in the cellulose strand introduced by the endocellulases, thusamplifying the single initiating reaction of the endocellulases.

Trichoderma reesei and Clostridium thermocellum are well-characterizedcellulose-utilizing organisms (Merino and Chemy, 2007; Bayer et al.,2008; Wilson, 2011). T. reesei is a slow-growing eukaryote fungus thatsecretes enzymes containing glycoside hydrolase (GH) domains fused tocarbohydrate binding domains, while C. thermocellum is a strictlyanaerobic prokaryote that predominantly assembles GHs andcarbohydrate-binding molecules (CBMs) into a large complex called thecellulosome. Enzymes from these free-living organisms cleavepolysaccharides using general acid-base catalyzed hydrolytic reactions(Vuong and Wilson, 2010). Moreover, fungal and microbial communitiesassociated with termites (Scharf et al., 2011) shipworms (Luyten et al.,2006), and rumen (Hess et al., 2011) contribute these types ofhydrolytic enzymes to their respective anaerobic niches.

Some free-living aerobes such as Cellvibrio japonicus (Ueda 107) (DeBoyet al., 2008), Streptomyces (Schlochtermeier et al., 1992; Wilson, 1992;Forsberg et al., 2011), Thermoascus aurantiacus (Langston et al., 2011;Quinlan et al., 2011) and Serratia marcescens (Vaaje-Kolstad et al.,2010) also grow on biomass polysaccharides. Recent work with some ofthese organisms has identified that the structurally related fungal GH61(Langston et al., 2011; Quinlan et al., 2011) and bacterial CBM33(Forsberg et al., 2011) families of proteins catalyze a previouslyunrecognized oxidative breakage of glycosidic bonds. This reaction isthought to be an endo-cleavage, with the oxidation reaction yieldinggluconate and keto-sugars instead of the typically observed reducing andnon-reducing sugars obtained from hydrolytic cellulases.

Actinobacteria in the genus Streptomyces are an ecologically importantgroup, especially in soil environments, where they are considered to bevital players in the decomposition of cellulose and other biomasspolymers (Cantarel et al., 2009; Crawford et al., 1978; Goodfellow andWilliams, 1983; McCarthy and Williams, 1992). Streptomyces are able toutilize a wide range of carbon sources, form spores when resources aredepleted, and produce antimicrobial secondary metabolites to reducecompetition (Goodfellow and Williams, 1983; Schlatter et al., 2009).

Although a large number of Streptomyces species can grow on biomass,only a small percentage (14%) have been shown to efficiently degradecrystalline cellulose (Wachinger, Bronnenmeier, et al., 1989).Furthermore, the secreted cellulolytic activities of only a few specieshave been biochemically characterized, and still fewer species have beenexamined to identify key biomass degrading enzymes (Ishaque andKluepfel, 1980; Semedo et al., 2004). Streptomyces reticuli is one ofthe best-studied cellulose- and chitin-degrading soil-dwellingStreptomyces; functional analyses of several important cellulases andother hydrolytic enzymes have been reported (Wachinger, Bronnenmeier, etal., 1989; Schlochtermeier, Walter, et al., 1992; Walter and Schrempf,1996).

Furthermore, polysaccharide monooxygenase (PMO) activity with cellulosewas identified using the CBM33 protein from Streptomyces coelicolor(Forsberg, et al., 2011), which suggests Streptomyces may use bothhydrolytic and oxidative enzymes to deconstruct biomass. With thetremendous amount of sequence data collected in the past few years, anddespite the view that Streptomyces make important contributions tocellulose degradation in the soil, genome-wide analyses of cellulolyticStreptomyces have not been reported.

In addition to their putative roles in carbon cycling in the soil,Streptomyces may also potentiate biomass deconstruction in insectsthrough symbiotic associations (Bignell, Anderson, et al., 1991; Pastiand Belli, 1985; Pasti, Pometto, et al., 1990; Schafer, et al., 1996).Recent work has identified cellulose degrading Streptomyces associatedwith the pine-boring woodwasp Sirex noctilio, including Streptomyces sp.SirexAA-E (ActE) (Adams, et al., 2011). S. noctilio is a highlydestructive wood-feeding insect that is found throughout forests inEurasia and North Africa and is spreading invasively in North Americaand elsewhere (Bergeron, et al., 2011). While the wasp itself does notproduce cellulolytic enzymes, evidence supports the role of a symbioticmicrobial community that secretes biomass-degrading enzymes tofacilitate nutrient acquisition for developing larvae in the pine tree(Kukor and Martin, 1983).

The white rot fungus, Amylostereum areolatum, is the best-describedmember of this community, and the success of Sirex infestations isthought to arise from the insect's association with this cellulolyticfungal mutualist. However, work with pure cultures has suggested thatActE and other Sirex-associated Streptomyces are more cellulolytic thanA. areolatum (Adams, et al., 2011).

Optimal activity in the CBM33 enzymes apparently requires the additionof a transition metal ion such as Cu(II), Fe(III), or Mn(II) and anexternal reducing agent. In the laboratory, the reducing agent can beprovided by ascorbate. In natural systems, the reducing function is mostlikely provided by another redox active protein such as cellobiosedehydrogenase (Langston et al., 2011; Quinlan et al., 2011) or someother presently unknown protein.

Needed in the art are improved compositions and organisms for digestionof lignocellulosic materials.

BRIEF SUMMARY

The invention relates generally to methods and compositions fordigesting lignocellulosic material and more particularly to methods thatinvolve exposing the material to secretome derived from Streptomyces sp.ActE.

In a first aspect, the present invention is summarized as a method ofdigesting a lignocellulosic material comprising the step of exposing thematerial to an effective amount of Streptomyces sp. ActE secretomepreparation such that at least partial lignocellulosic digestion occurs.

In some embodiments of the first aspect, the preparation is asupernatant preparation obtained from a Streptomyces sp. ActE culture.In some embodiments of the first aspect, the preparation is obtainedfrom Streptomyces sp. ActE grown on a substrate wherein at least 40%,preferably 85%, of Streptomyces sp. ActE's carbon source in thesubstrate is derived from a material selected from the group consistingof cellulose, cellulose/hemicelluloses mixture, hemicelluloses, xylan,non-wood biomass, wood biomass and chitin. In some embodiments of thefirst aspect, the lignocellulosic material is selected from the groupconsisting of materials that comprise at least 75% cellulose,cellulose/hemicelluloses, xylose, biomass and chitin.

In a second aspect, the present invention is summarized as a purifiedpreparation comprising the Streptomyces sp. ActE secretome.

In some embodiments of the second aspect, the preparation is asupernatant preparation obtained from a Streptomyces sp. ActE culture.In some embodiments of the second aspect, Streptomyces sp. ActE is grownon a substrate wherein at least 40%, preferably 85%, of Streptomyces sp.ActE's carbon source in the substrate is derived from a materialselected from the group consisting of cellulose,cellulose/hemicelluloses mixture, hemicelluloses, xylan, non-woodbiomass, wood biomass and chitin.

In a third aspect, the present invention is summarized as a compositionuseful for digesting lignocellulosic material comprising SActE_(—)0237(GH6) (SEQ ID NOs:1 and 17) gene or expression product thereof.

In a fourth aspect, the present invention is summarized as a compositionuseful for digesting lignocellulosic material comprising SActE_(—)0236(GH48) (SEQ ID NOs:2 and 18) gene or expression product thereof.

In a fifth aspect, the present invention is summarized as a compositionuseful for digesting lignocellulosic material comprising SActE_(—)3159(CBM33) (SEQ ID NOs:3 and 19) gene or expression product thereof.

In a sixth aspect, the present invention is summarized as a compositionuseful for digesting lignocellulosic material comprising SActE_(—)0482(GH5) (SEQ ID NOs:4 and 20) gene or expression product thereof.

In a seventh aspect, the present invention is summarized as acomposition useful for digesting lignocellulosic material comprisingSActE_(—)0265 (GH10) (SEQ ID NOs:5 and 21) gene or expression productthereof.

In a eighth aspect, the present invention is summarized as a compositionuseful for digesting lignocellulosic material comprising SActE_(—)2347(GH5) (SEQ ID NOs:6 and 22) gene or expression product thereof.

In a ninth aspect, the present invention is summarized as a compositionuseful for digesting lignocellulosic material comprising SActE_(—)0237(GH6) (SEQ ID NOs: 1 and 17), SActE_(—)0236 (GH48) (SEQ ID NOs: 2 and18), SActE_(—)3159 (CBM33) (SEQ ID NOs: 3 and 19), SActE_(—)0482 (GH5)(SEQ ID NOs: 4 and 20) and gene or expression product thereof.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth,and ninth aspects, the composition is optimized for celluloseutilization. In these embodiments the composition can additionallycomprise at least one member selected from SActE_(—)0265 (GH10) (SEQ IDNOs: 5 and 21) and SActE_(—)2347 (GH5) (SEQ ID NOs: 6 and 22) genes orexpression products thereof. In a preferred embodiment, the compositioncomprises at least three or four of the genes or expression products.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth,and ninth aspects, the composition is optimized for xylan release. By“release,” we mean degradation, such as hydrolysis, and release of animportant or desired product. In these embodiments the composition canadditionally comprise at least one member selected from SActE_(—)0265(GH10) (SEQ ID NOs: 5 and 21), SActE_(—)0358 (GH11) (SEQ ID NOs: 8 and24), SActE_(—)0357 (CE4) (SEQ ID NOs: 7 and 23), SActE_(—)5978 (PL1)(SEQ ID NOs: 16 and 32) and SActE_(—)5230 (xylose isomerase) (SEQ IDNOs:33 and 48) genes or expression products thereof. In a preferredembodiment, the composition comprises at least three or four of thegenes or expression products.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth,and ninth aspects, the composition is optimized for chitin release. Inthese embodiments the composition can additionally comprise at least onemember selected from SActE_(—)4571 (GH18) (SEQ ID NOs:34 and 49),SActE_(—)2313 (CBM33) (SEQ ID NOs:35 and 50), SActE_(—)4246 (GH18), (SEQID NOs:36 and 51) SActE_(—)3064 (GH19) (SEQ ID NOs:37 and 52), andSActE_(—)5764 (GH18) (SEQ ID NOs:38 and 53) genes or expression productsthereof. In a preferred embodiment, the composition comprises at leastthree or four of the genes or expression products.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth,and ninth aspects, the composition is optimized for biomass degradation.In these embodiments the composition can additionally compriseSActE_(—)5457 (GH46) (SEQ ID NOs: 14 and 30) gene or expression productsthereof.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth,and ninth aspects, the composition is optimized for mannan release. Inthese embodiments the composition can additionally compriseSactE_(—)2347 (GH5) (SEQ ID NOs: 6 and 22) gene or expression productsthereof.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth,and ninth aspects, the composition is optimized for beta-1,3-glucanrelease. In these embodiments the composition can additionally compriseat least one member selected from SActE_(—)4755 (GH64) (SEQ ID NOs:13and 29) and SActE_(—)4738 (GH16) (SEQ ID NOs:12 and 28) genes orexpression products thereof. In a preferred embodiment, the compositioncomprises both of the genes or expression products.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth,and ninth aspects, the composition is optimized for pectin cleavage. Inthese embodiments the composition can additionally compriseSActE_(—)1310 (PL3) (SEQ ID NOs:9 and 25) gene or expression productsderived thereof.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth,and ninth aspects, the composition is optimized for alginate release. Inthese embodiments the composition can additionally compriseSActE_(—)4638 (SEQ ID NOs:11 and 27) gene or expression products derivedthereof.

In some embodiments of the third, fourth, fifth, sixth, seventh, eighth,and ninth aspects, the composition is optimized for galactose release.In these embodiments the composition can additionally compriseSactE_(—)5647 (GH87) (SEQ ID NOs:15 and 31) gene or expression productsderived thereof.

In a tenth aspect, the present invention is summarized as a compositionuseful for xylan degradation comprising SActE_(—)0265 (GH10) (SEQ IDNOs:5 and 21) and SActE_(—)0358 (GH11) (SEQ ID NO:8 and 24) gene orexpression products thereof.

In some embodiments of the tenth aspect, the composition additionallycomprises SActE_(—)0265 (GH10) (SEQ ID NOs:5 and 21), SActE_(—)0358(GH11) (SEQ ID NOs:8 and 24), SActE_(—)0357 (CE4) (SEQ ID NOs:7 and 23),SActE_(—)5978 (PL1) (SEQ ID NOs:16 and 32), and SActE_(—)5230 (xyloseisomerase) (SEQ ID NOs:33 and 48) genes or expression products thereof.In a preferred embodiment, the composition comprises at least three orfour of the genes or expression products.

In an eleventh aspect, the present invention is summarized as acomposition useful for biomass degradation comprising SActE_(—)0237(GH6) (SEQ ID NOs:1 and 17), SActE_(—)0482 (GH5) (SEQ ID NOs:4 and 20),SActE_(—)3159 (CBM33) (SEQ ID NOs:3 and 19), SActE_(—)0236 (GH48) (SEQID NOs:2 and 18), SActE_(—)3717 (GH9) (SEQ ID NOs:10 and 26),SActE_(—)0265 (GH10) (SEQ ID NOs:5 and 21), SActE_(—)0358 (GH11) (SEQ IDNOs:8 and 24), SActE_(—)2347 (GH5) (SEQ ID NOs:6 and 22) andSActE_(—)1310 (PL3) (SEQ ID NOs:9 and 25) genes or expression productsthereof. In a preferred embodiment, the composition comprises at leastthree or four of the genes or expression products.

In a twelfth aspect, the present invention is summarized as acomposition useful for cellulose degradation comprising SActE_(—)0237(GH6) (SEQ ID NOs:1 and 17), SActE_(—)0482 (GH5) (SEQ ID NOs:4 and 20),SActE_(—)3159 (CBM33) (SEQ ID NOs:3 and 19), SActE_(—)0236 (GH48) (SEQID NOs:2 and 18), SActE_(—)2347 (GH5) (SEQ ID NOs:6 and 22), andSActE_(—)0265 (GH10) (SEQ ID NOs:5 and 21) genes or expression productsthereof. In a preferred embodiment, the composition comprises at leastthree or four of the genes or expression products.

In a thirteenth aspect, the present invention is summarized as a methodfor digesting a lignocellulosic material, comprising exposing thematerial to a sufficient amount of a composition of any one of the thirdto eighth aspects of the invention, wherein the exposed material is atleast partially digested.

In a fourteenth aspect, the present invention is summarized as apurified preparation of Streptomyces sp. ActE, wherein the Streptomycessp. ActE has been grown on a substrate wherein at least 40%, preferably85%, of Streptomyces sp. ActE's carbon source in the substrate isderived from a material selected from the group consisting of cellulose,cellulose/hemicelluloses mixture, hemicelluloses, xylan, non-woodbiomass, wood biomass, and chitin.

In a fifteenth aspect, the present invention is summarized as a purifiedpreparation of Streptomyces sp. ActE, wherein the Streptomyces sp. ActEhas been grown on a substrate wherein at least 40%, preferably 85%, ofStreptomyces sp. ActE's carbon in the substrate is derived frompretreated lignocellulosic material.

In some embodiments of the fifteenth aspect, the pretreated material hasbeen exposed to pretreatment selected from the group consisting of acidhydrolysis, steam explosion, ammonia fiber expansion (AFEX),organosolve, sulfite pretreatment to overcome recalcitrance oflignocellulose (SPORL), ionic liquids, metal-catalyzed hydrogenperoxide, alkaline wet oxidation and ozone pretreatment. In someembodiments of the fifteenth aspect, the pretreated material is wood.

These and other features, objects, and advantages of the presentinvention will become better understood from the description thatfollows. In the description, reference is made to the accompanyingdrawings, which form a part hereof and in which there is shown by way ofillustration, not limitation, embodiments of the invention. Thedescription of preferred embodiments is not intended to limit theinvention to cover all modifications, equivalents and alternatives.Reference should therefore be made to the claims recited herein forinterpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The present invention will be better understood and features, aspectsand advantages other than those set forth above will become apparentwhen consideration is given to the following detailed descriptionthereof. Such detailed description makes reference to the followingdrawings, wherein:

FIG. 1 is a set of pictures showing growth of ActE in minimal mediumcontaining filter paper as the sole carbon source. (A) Growth of ActE,Streptomyces coelicolor, and Streptomyces griseus in minimal medium for7 days at 30° C. and pH 6.9. The expanded image shows small colonies ofS. coelicolor and S. griseus forming on the surface of the paper. (B)Growth of ActE and Trichoderma reesei Rut-C30 for 7 days at 30° C. andpH 6.0.

FIG. 2 is a set of graphs demonstrating reactions of ActE secretomes andSPEZYME-CP. (A) HPLC of sugars released from cellulose (1, cellotriose;2, cellobiose; 3, glucose) and quantification of glucose equivalent(insert). (B) Reducing sugars released from xylan and mannan by thesecretomes of ActE grown on cellulose and xylan. (C) Total reducingsugar released from ionic liquid-switchgrass (IL-SG) or AFEX-switchgrass(AFEX-SG) in reactions of the ActE cellulose, AFEX-SG, and IL-SGsecretomes and Spezyme CP. Data represent the mean±s.d. from threeexperiments; * indicates P<0.01 compared with SPEZYME CP.

FIG. 3 is a table illustrating composition of ActE secretomes identifiedby LC-MS/MS. (A) Carbohydrate Active Enzyme (CAZy) genes account for2.6% of the 6357 predicted protein-coding sequences in the ActE genome.(B) Identity of most abundant proteins in the cellulose secretomeproteins is sorted according to decreasing spectral counts (accountingfor 95% of total spectral counts); corresponding spectral counts fromother secretomes are also shown.

FIG. 4 is a systematic diagram showing genome-wide changes in expressionduring growth of ActE on AFEX-treated switchgrass (AFEX-SG) versusglucose. Nodes are genes (circles) or KEGG/CAZy functional categories(yellow triangles); edges indicate that the gene belongs to theindicated functional group as defined by either KEGG or CAZy analysis.Gene node sizes reflect expression intensity determined by microarrayfrom growth on AFEX-SG as a log₂ ratio, where the genome-wide averagetranscriptional intensity was ˜10.5 for both substrates. Node colorsrepresent expression changes as the log₂ ratio of AFEX-SG/glucosetranscript intensities.

FIG. 5 is a diagram with a table showing expression of ActE CAZy geneson various carbon sources. (A) Hierarchical clustering of expression for167 CAZy genes from the ActE genome during growth on the indicatedsubstrates. (B) Identity of CAZy genes with distinct changes inexpression observed in group 1 CAZy genes during growth in differentcarbon sources.

FIG. 6 is a set of scanning electron microscopy (SEM) images showingActE grown on different carbon sources including glucose, cellulose,xylan, switchgrass, ammonia fiber expansion-treated switchgrass(AFEX-SG) and ionic liquid-treated switchgrass (IL-SG). ActE cells weregrown in minimum medium with the indicated substrate as a sole carbonsource for 7 days at 30° C. The scale bar indicates 5 μm.

FIG. 7 is a set of graphs demonstrating fractionation of the ActEcellulose secretome and assays of reactions with differentpolysaccharides. (A) Anion exchange chromatography was performed usingthe ActE cellulose secretome, and fractions were collected and analyzedby SDS-PAGE. Lowercase letters indicate protein identified by MALDI-TOFMS shown in FIG. 17. (B) Results from hydrolysis assays for reactionwith filter paper (FP), xylan, mannan and beta-1,3 glucan as detected byDNS assay of each fraction. The percentage reactivity relative to themaximum activity observed for each substrate is shown. Error barsindicate the standard deviation, with n=3 for technical replicates.

FIG. 8 a set of diagrams showing temperature and pH profiles of the ActEsecretome obtained from growth on AFEX-treated corn stover. (A) Theeffect of temperature on the deconstruction of AFEX-treated switchgrass(AFEX-SG) and ionic liquid-treated switchgrass (IL-SG). The relativeactivity of the ActE secretome was compared to the maximal ratesdetermined for reaction with AFEX-SG (blue star), and IL-SG (red star)at pH 6.0. (B) The effect of pH on the AFEX-SG and IL-SG deconstructionactivities in the indicated ActE secretomes. The maximal rates observedfor AFEX-SG and IL-SG were at pH 7.0 (blue star) and pH 8 (red star),respectively. Reactions were carried out at 40° C. and the 0.1 M buffersused were citrate (pH 4.5), phosphate (pH 6-8), CHES (pH 9-10), and CAPS(pH 11). The reaction was performed for 20 h and the reducing sugarcontent was measured by DNS assay.

FIG. 9 is a systematic diagram showing genome-wide changes in expressionduring growth of ActE on substrate cellobiose versus glucose visualizedas a Cytoscape interaction network. Nodes are genes (circles) orKEGG/CAZy functional categories (yellow triangles); edges indicate thatthe gene belongs to the indicated functional group as defined by eitherKEGG or CAZy analysis. Gene node sizes reflect expression intensitydetermined by microarray from growth on substrate as a log 2 ratio. Nodecolors represent expression changes as the log 2 ratio ofsubstrate/glucose transcript intensities, where the genome-wide averagetranscriptional intensity was ˜10.5 for both substrate and glucose.Transcripts with less than two-fold changes in expression intensity arecolored white; transcripts with greater than two-fold increase inexpression intensity during growth on substrate are shown as a redgradient; transcripts with greater than two-fold increase in expressionintensity during growth on glucose are shown as a blue gradient.

FIG. 10 is a systematic diagram showing genome-wide expression changesfor growth on the substrate cellulose versus glucose visualized as aCytoscape interaction network. Other information is the same as thatdescribed in FIG. 9.

FIG. 11 is a systematic diagram showing genome-wide expression changesfor growth on the substrate xylan versus glucose visualized as aCytoscape interaction network. Other information is the same as thatdescribed in FIG. 9.

FIG. 12 is a systematic diagram showing genome-wide expression changesfor growth on the substrate switchgrass versus glucose visualized as aCytoscape interaction network. Other information is the same as thatdescribed in FIG. 9.

FIG. 13 is a systematic diagram showing genome-wide expression changesfor growth on the substrate IL-treated switchgrass versus glucosevisualized as a Cytoscape interaction network. Other information is thesame as that described in FIG. 9.

FIG. 14 is a systematic diagram showing genome-wide expression changesfor growth on the substrate chitin versus glucose visualized as aCytoscape interaction network. Other information is the same as thatdescribed in FIG. 9.

FIG. 15 is a diagram with a table showing expression of 167 predictedCAZy genes in ActE, highlighting group 2 genes. These genes showed nosignal above the average genomic expression intensity (log 2=10.5). (A)Clustering of genes with similar expression profiles. (B) Additionalinformation on group 2 genes including expression profile, SACTE_locusID, CAZy family, and annotated function.

FIG. 16 is a diagram with a table showing expression of 167 predictedCAZy genes in ActE, highlighting group 3 genes. (A) Clustering of geneswith similar expression profiles. (B) Additional information on group 3genes including expression profile, SACTE_locus ID, CAZy family, andannotated function.

FIG. 17 is a table illustrating proteins separated by ion exchangechromatography and identified by mass spectrometry.

FIG. 18 is a table showing spectra count of proteins identified on eachsubstrate, where top 95% spectra covered were highlighted green, lightpurple, purple, blue, orange, pink, light blue and yellow on glucose,cellobiose, cellulose, xylan, switchgrass, AFEX-SG, IL-SG and chitin,respectively.

FIG. 19 shows the nucleic acid sequences of the ActE genes.

FIG. 20 shows the amino acid sequences of the ActE genes.

FIG. 21 is a graph illustrating a comparison of specific activities ofStreptomyces sp. ActE secretomes with SPEZYME CP. FIG. 21A depictsrelative specific activity of ActE secretomes prepared from growth oncellulose or xylan and SPEZYME CP (100%) for reducing sugar release fromxylan or mannan. FIG. 21B depicts relative activity (pH 6.0, 40° C.) ofActE cellulose secretome and CelLcc_CBM3a, an engineered C. thermocellumendo/exoglucanase, compared to SPEZYME CP. Total amounts of proteinincluded in all reactions were equivalent.

FIG. 22 illustrates nucleotide and amino acid sequence of CelLcc_CBM3a.Construct described in US Patent Application Publication No.:US2010/037094 (Fox and Elsen).

FIG. 23 is a graph illustrating SDS-PAGE of Streptomyces sp. ActEsecretomes obtained from growth on minimal medium containing differentsubstrates (SG, switchgrass; CS, corn stover; UBLPKP, unbleachedlodgepole pine kraft pulp; BSKP, bleached spruce kraft pulp; LP-SPORL,lodgepole pine pretreated by sulfite pretreatment to overcomerecalcitrance of lignocellulose (SPORL)). Culture secretomes wereseparated after 7 days of growth at 30° C. by centrifugation andconcentrated by ultrafiltration. Sample loading was normalized to totalprotein. The identities of proteins were determined from samplesextracted from the SDS-PAGE gel. Among the 162 proteins accounting for95% of spectral counts from the glucose secretome, most wereintracellular proteins originating from cell lysis during growth, andwere not detected in the polysaccharide secretomes.

FIG. 24 is a graph illustrating SDS-PAGE of time-dependent changes inthe Streptomyces sp. ActE secretome obtained from growth on minimalmedium containing cellulose. Culture secretomes were collected after 7days by centrifugation and concentrated by ultrafiltration. Theconcentrated secretomes were incubated at 25° C. for the indicated timesand analyzed. Protein bands with time-dependent decrease in intensitywere excised from the gel and identified by LC-MS/MS.

FIG. 25 illustrates synergy of recombined fractions from ion exchangechromatography. All reactions were prepared to contain the same totalamount of protein.

FIG. 26 is a set of graphs illustrating mannanase activity demonstratedin fractions containing various naturally truncated versions ofSACTE_(—)2347 (GH5). FIGS. 26A-B depict proteins found in previousassayed fractions. FIG. 26C depicts Coomassie Blue staining of 12%polyacrylamide gel (PAGE) separation of different mannanase isoforms.Three polypeptide bands corresponding to SACTE_(—)2347 (GH5) withmolecular masses of ˜57, ˜45, and ˜37 kDa. FIG. 26D depicts a zymogramperformed in the presence of 0.5% mannan. The strong clearing zone infraction F1 associated with the ˜37 kDa isoform demonstrates how sizereduction can increase the specific activity of a protein.

FIG. 27 is a set of graphs illustrating ion exchange fractionation ofStreptomyces sp. ActE secretome. FIG. 27A depicts an SDS-PAGE analysisof the fractionation of an ActE secretome by ion exchangechromatography. FIG. 27B depicts catalytic assays of the separatefractions at 40° C. for 20 h in 0.1 M phosphate buffer, pH 6.0, showingdifferent enzymes are capable of reacting with xylan, mannan, andcellulose. The reactivity of fractions marked with stars is alsodescribed in FIG. 25A.

FIG. 28 is a SDS-PAGE graph and a list illustrating mass spectralassignment of polypeptides from the Streptomyces sp. ActE secretomeseparated by ion exchange chromatography. FIG. 28A depicts an SDS PAGEof separated fractions annotated with identities of polypeptidesdetermined by LC-MS analysis. FIG. 28B depicts information on theidentified proteins including gene locus, function, CAZy GH and CBMassignments, number of amino acid (AA) residues, and best BLAST resultfor relationship to another known enzyme. The reactivity of fractionsmarked with stars is also described in FIG. 25A.

FIG. 29 is a SDS-PAGE graph and a table that demonstrates the existenceof xylanases from Streptomyces sp. ActE. Five ActE proteins wereproduced using cell-free translation as described in US PatentApplication Publication No.: US2010/037094 (Fox and Elsen). FIG. 29Adepicts a stain-free gel image of proteins produced by wheat germcell-free translation (indicated by asterisks). FIG. 29B depicts asummary of protein information, expression and secretion data, anddiagnostic assay results. Small molecule assays (MUG, methylumbelliferylglucoside; MUC, methylumbelliferyl cellobioside; MUM, methylumbelliferylmannoside and MUX2, methylumbelliferyl xylobioside) were performed in0.1 M phosphate buffer, pH 6.0, at 30° C. SACTE_(—)0265 andSACTE_(—)0358, highly expressed and secreted proteins during growth onxylan, are confirmed by these assays to be xylanases. Results from threeother non-secreted ActE enzymes are provided as controls.

FIG. 30 is a graph illustrating quantification of total secreted proteinobtained from Streptomyces sp. ActE grown on different substrates(AFEX-CS, AFEX corn stover; UBLPKP, unbleached lodgepole pine kraftpulp; BSKP, bleached spruce kraft pulp; LP-SPORL, lodgepole pinepretreated by SPORL).

FIG. 31 is a graph illustrating the temperature versus activity profileof the Streptomyces sp. ActE secretome obtained from growth oncellulose. Hydrolysis activities were measured by DNS assay. Greaterthan 80% of maximal rates for cellulase and mannase activity wereobserved at the range of 31-43° C., while greater than 80% of maximalrate for xylanase activity was observed in the range of 35-59° C.

FIG. 32 is a graph illustrating the pH versus activity profile of theStreptomyces sp. ActE secretome obtained from growth on cellulose. Themaximal rate was observed at approximately pH 6. Buffers used in thisstudy were 0.1 M citrate (pH 4.5), phosphate (pH 6-8), CHES (pH 9-10)and CAPS (pH 11).

FIG. 33 is a SDS-PAGE graph illustrating ActE induction in mediumcontaining as little as 20% cellulose.

FIG. 34 is a set of Venn diagrams representing 95% of total proteinsidentified in LC-MS/MS analyses generated using VennMaster-0.37.5(Kestler et al., 2008). FIG. 9A depicts secretomes obtained from growthon glucose, Sigmacell™, and xylan. FIG. 9B depicts secretomes obtainedfrom growth on switchgrass, ammonia fiber expansion (AFEX)-SG, andIL-SG. For clarification, glucose ∩ Sigmacell)=4 represents theintersection of the two sets, while glucose/(Sigmacell

xylan)=117 represents the proteins uniquely associated with growth onglucose as compared to Sigmacell. Other results are interpreted in asimilar manner.

While the present invention is susceptible to various modifications andalternative forms, exemplary embodiments thereof are shown by way ofexample in the drawings and are herein described in detail. It should beunderstood, however, that the description of exemplary embodiments isnot intended to limit the invention to the particular forms disclosed,but on the contrary, the intention is to cover all modifications,equivalents and alternatives falling within the spirit and scope of theinvention as defined by the appended claims.

DESCRIPTION OF EXEMPLARY EMBODIMENTS In General

The present invention comprises many embodiments. In one embodiment, theinvention is a method of digesting a lignocellulosic material,comprising the step of exposing the material to an effective amount ofStreptomyces sp. ActE secretome preparation such that at least partiallignocellulosic digestion occurs. In one embodiment of that method, thepreparation is a supernatant preparation obtained from a Streptomycessp. ActE culture. In another embodiment of that method, the preparationis obtained from Streptomyces sp. ActE grown on a substrate wherein atleast 40%, preferably 85%, of Streptomyces sp. ActE's carbon source inthe substrate is derived from a material selected from the groupconsisting of cellulose, cellulose/hemicelluloses mixture,hemicelluloses, xylan, non-wood biomass, wood biomass, and chitin. Inanother embodiment of that method, the lignocellulosic material isselected from the group consisting of materials that comprise at least75% cellulose, cellulose/hemicelluloses, xylose, biomass and chitin.

In one embodiment, the invention is a purified preparation comprisingthe Streptomyces sp. ActE secretome. In one embodiment, the preparationis a supernatant preparation obtained from a Streptomyces sp. ActEculture. In another embodiment of the preparation, Streptomyces sp. ActEis grown on a substrate wherein at least 40%, preferably 85%, ofStreptomyces sp. ActE's carbon source in the substrate is derived from amaterial selected from the group consisting of cellulose,cellulose/hemicelluloses mixture, hemicelluloses, xylan, non-woodbiomass, wood biomass, and chitin.

In one embodiment, the invention is a composition useful for digestinglignocellulosic material comprising one gene or expression productthereof selected from the group consisting of SActE_(—)0237 (GH6) (SEQID NOs:1 and 17), SActE_(—)0236 (GH48) (SEQ ID NOs:2 and 18),SActE_(—)3159 (CBM33) (SEQ ID NOs:3 and 19), SActE_(—)0482 (GH5) (SEQ IDNOs:4 and 20), SActE_(—)0265 (GH10) (SEQ ID NOs:5 and 21), andSActE_(—)2347 (GH5) (SEQ ID NOs:6 and 22) genes or expression productsthereof. In one embodiment, the composition additionally comprises atleast one member selected from the group consisting of SActE_(—)0357(CE4) (SEQ ID NOs:7 and 23), SActE_(—)0358 (GH11) (SEQ ID NOs:8 and 24),SActE_(—)1310 (PL3) (SEQ ID NOs:9 and 25), SActE_(—)3717 (GH9) (SEQ IDNOs:10 and 26), SActE_(—)4638 (SEQ ID NOs:11 and 27), SActE_(—)4738(GH16) (SEQ ID NOs:12 and 28), SActE_(—)4755 (GH64) (SEQ ID NOs:13 and29), SActE_(—)5457 (GH46) (SEQ ID NOs:14 and 30), SActE_(—)5647 (GH87)(SEQ ID NOs:15 and 31), and SActE_(—)5978 (PL1) (SEQ ID NOs:16 and 32)genes or expression products derived thereof.

In one embodiment, the invention is a composition useful for cellulosedegradation comprising SActE_(—)0236 (GH48) (SEQ ID NOs:2 and 18),SActE_(—)3159 (CBM33) (SEQ ID NOs:3 and 19), SActE_(—)0482 (GH5) (SEQ IDNOs:4 and 20) and SActE_(—)0237 (GH6) (SEQ ID NOs:1 and 17) genes orexpression product thereof. In one embodiment, the compositionadditionally comprises at least one member selected from the groupconsisting of SActE_(—)0357 (CE4) (SEQ ID NOs:7 and 23), SActE_(—)0358(GH11) (SEQ ID NOs:8 and 24), SActE_(—)1310 (PL3) (SEQ ID NOs:9 and 25),SActE_(—)3717 (GH9) (SEQ ID NOs:10 and 26), SActE_(—)4638 (SEQ ID NOs:11and 27), SActE_(—)4738 (GH16) (SEQ ID NOs:12 and 28), SActE_(—)4755(GH64) (SEQ ID NOs:13 and 29), SActE_(—)5457 (GH46) (SEQ ID NOs:14 and30), SActE_(—)5647 (GH87) (SEQ ID NOs:15 and 31), and SActE_(—)5978(PL1) (SEQ ID NOs:16 and 32) genes or expression products derivedthereof.

In one embodiment, the invention is a method for digesting alignocellulosic material, comprising exposing the material to asufficient amount of a composition of any combinations of genes orexpression products derived thereof as disclosed above, wherein theexposed material is at least partially digested.

In one embodiment, the invention is a purified preparation ofStreptomyces sp. ActE, wherein the Streptomyces sp. ActE has been grownon a substrate wherein at least 40%, preferably 85%, of Streptomyces sp.ActE's carbon source in the substrate is derived from a materialselected from the group consisting of cellulose,cellulose/hemicelluloses mixture, hemicelluloses, xylan, non-woodbiomass, wood biomass and chitin.

In one embodiment, the invention is a purified preparation ofStreptomyces sp. ActE, wherein the Streptomyces sp. ActE has been grownon a substrate wherein at least 40%, preferably 85%, of Streptomyces sp.ActE's carbon in the substrate is derived from pretreatedlignocellulosic material. In one embodiment of the preparation, thepretreated material has been exposed to pretreatment selected from thegroup consisting of acid hydrolysis, steam explosion, ammonia fiberexpansion (AFEX), organosolve, sulfite pretreatment to overcomerecalcitrance of lignocellulose (SPORL), ionic liquids (IL),metal-catalyzed hydrogen peroxide treatment, alkaline wet oxidation andozone pretreatment. In another embodiment of the preparation, thepretreated material is wood.

Specific Embodiments

Applicants have been interested in insects that utilize plant biomassand their associated microbial and fungal communities. Sirex noctilio, awood boring wasp, is found in pine forests throughout Eurasia and NorthAfrica and is spreading throughout North America and elsewhere (Bergeronet al., 2011). Although the destructive nature of the Sirex infestationis generally considered to arise from a symbiotic relationship betweenS. noctilio and Amylostereum areolatum, a white rot basidiomycete (Kukorand Martin, 1983; Klepzig et al., 2009; Bergeron et al., 2011), the roleof cellulolytic microbes has not been previously considered in thecontext of the infestation or symbiosis. Streptomyces sp. SirexAA-E[Streptomyces sp. ActE, also referred to herein as “ActE” (Adams et al.,ISME J. 5:1321-1231, 2011)], was isolated from the ovipositor mycangiumof S. noctilio (Adams et al., 2011). Applicants hypothesized that ActEis inoculated into insect feeding tunnels upon infestation along withthe symbiotic fungus. Thus, Applicants were interested to learn how ActEmight contribute to the Sirex community.

The present invention will be more fully understood upon considerationof the following non-limiting Examples. All papers and patents disclosedherein are hereby incorporated by reference as if set forth in theirentirety.

As used herein, the term “ActE” refers to Streptomyces sp. SirexAA-E, asdescribed in Adams et al., ISME J. 5:1321-1231, 2011. A representativesample of Streptomyces sp. ActE has been deposited according to theBudapest Treaty for the purpose of enabling the present invention. Therepository selected for receiving the deposit is the American TypeCulture Collection (ATCC) having an address at 10801 UniversityBoulevard, Manassas, Va. USA, Zip Code 20110. The ATCC repository hasassigned the patent deposit designation PTA-12245 to the Streptomycessp. ActE strain.

As used herein, the term “secretome” refers to the plurality of secretedenzymes. For example, ActE secretome refers to the secreted enzymes fromStreptomyces sp. SirexAA-E.

As used herein, the term “lignocellulosic material” refers to anymaterial that is composed of cellulose, hemicellulose, and lignin,wherein the carbohydrate polymers (cellulose and hemicelluloses) aretightly bound to the lignin.

As used herein, the term “biomass” refers to a renewable energy source,and comprises biological material from living or recently livingorganisms. As an energy source, biomass can either be used directly, orconverted into other energy products such as biofuel. Biomass includesplant or animal matter that can be converted into fibers or otherindustrial chemicals, including biofuels. Industrial biomass can begrown from numerous types of plants, including miscanthus, switchgrass,hemp, corn, poplar, willow, sorghum, sugarcane, bamboo, and a variety oftree species, ranging from eucalyptus to oil palm (palm oil). Thus,biomass can include wood biomass and non-wood biomass.

The present invention has multiple embodiments. All embodiments arerelated to Applicants' discovery of improved lignocellulosic digestionand utilization using proteins and genes obtained from the Streptomycessp. ActE secretome.

ActE Isolates and Secretomes

Streptomyces sp. SirexAA-E may be isolated from ovipositor mycangia ofS. noctilio. In Adams, et al, S. noctilio were collected from apopulation in Pennsylvania, USA. Infested trees were cut and transportedto USDA Pest Survey, Detection, and Exclusion Lab in Syracuse, N.Y., USA(Zylstra et al. (2010) Agric. Forest. Entomol. in press). Four adultfemales and six larvae from the Pennsylvania population were sampled,and cultures of bacteria derived from these insect samples were screenedfor cellulose degradation.

Prior to sampling for bacteria, all insects were typically surfacesterilized in 95% ethanol for 1 minute and then rinsed twice in sterilephosphate-buffered saline (1×PBS). Larval guts and adult ovipositors andmycangia were removed surgically. These segments and the body wereground separately in 1 ml 1×PBS using a sterilized mortar and pestle. 50μl of three 100-fold dilutions of each insect part were plated ontoyeast and malt extract agar (Becton, Dickinson and Company, Sparks, Md.,USA), acidified yeast malt extract agar (for gut dissections only), 10%tryptic soy agar (Becton, Dickinson and Company, Sparks, Md., USA), andagar supplemented with chitin (MP Biomedicals, Solon, Ohio). Petridishes were stored at room temperature in darkness for at least threedays until visible colonies formed, except for Petri dishes with chitinagar that were stored for at least one month.

All isolates were typically screened for production of cellulolyticenzymes on carboxymethyl cellulose (CMC) (Teather R M, Wood P J (1982);incorporated herein by reference as if set forth in its entirety).Isolates that tested positive on CMC were then studied further. Assayson CMC, AFEX-treated corn stover at three pH levels, andmicrocrystalline cellulose were typically performed to assess growth anddegradation ability of each insect-derived bacterial isolate. Isolatescapable of degrading CMC were further analyzed genomically to identifyisolates with high Carbohydrate Active Enzyme (CAZy) content relative toone another and relative to known organisms. Streptomyces sp. ActE wasselected based on its CMC degradation and CAZy gene profile.

In one embodiment, secretomes from ActE would be used alone in a firstreaction to convert biomass into a hydrolyzed solution of sugars thatwould be used in a second reaction with a fermentation organism toconvert the sugars into usable biofuels. The first and second reactioncould occur simultaneously.

In a second embodiment, secretomes from ActE would be combined withsecretomes from other organisms, or with enzymes or enzyme compositions,such as Spezyme CP, to increase the activity of both preparations bysynergy of the enzymes contained in each preparation.

Preferably, the ActE secretomes would be prepared as supernatants fromActE cultures.

In one embodiment, the supernatant is prepared by centrifugation of theActE culture for 10 min at 3,000×g, which will pellet the remaininginsoluble polysaccharide and adhered ActE cells. The supernatantfraction is filter-sterilized, preferably using a 0.22 μm filter, inorder to remove any remaining cells. The supernatant is concentrated,preferably using a 3 kDa cut-off ultrafiltration membrane. Theconcentration of total protein is determined by Bradford assay(Bradford, 1976). In one preferred embodiment, the proteomic compositionof the ActE secretome is that described in FIG. 3 or FIG. 18.

The secretomes obtained from growth on specific lignocellulosicmaterials, such as cellulose, xylan, cellulosic hemi-cellulosic biomass,and chitin, will have distinct compositions of individual enzymes andalso distinct reactivity with different polysaccharides. The cellulosichemi-cellulosic biomass may be non-wood biomass or wood biomass. Forexample, the secretome prepared from ActE grown on cellulose has uniqueenzymes and enhanced reactivity with cellulose and mannan. Also, thesecretome prepared from ActE grown on xylan possesses high xylandegradation activity, whereas the secretome from ActE grown on chitinpossesses uniquely high chitin degradation activity. Example A disclosesthe specific secretomes.

When ActE is grown on switchgrass, AFEX-pretreated switchgrass or ionicliquid pretreated switchgrass, the secretome has a protein compositionthat partially matches that obtained from growth on either cellulose orxylan. However, switchgrass, AFEX-pretreated switchgrass or ionic liquidpretreated switchgrass elicit the appearance of new proteins in thesecretome that enhance the degradative ability of the secretome for theplant biomass materials. Applicants envision that the present inventionwould also apply to other pretreatment methods comprising acidhydrolysis, steam explosion, organosolve, sulfite pretreatment toovercome recalcitrance of lignocellulose (SPORL), metal-catalyzedhydrogen peroxide treatment, alkaline wet oxidation and ozonepretreatment.

The inventors' preliminary data shows synergistic filter paper degradingactivity between the ActE secretome and other cellulases from adifferent organism. Also, addition of a beta-glucosidase to thesecretome helps to break down the oligosaccharides (e.g., cellotetraose,cellotriose and cellobiose) released from filter paper into simplersugars.

Preferably, the secretome would be prepared as a concentrated solutionby ultrafiltration. The concentrated material would be mixed with thesubstrate at weight percentages varying from 0.1% to 20% w/w, with theremainder of the solution containing a buffer substance that controlspH. Trace metals would be added to the reaction. The material would beincubated at the appropriate temperature to allow the reaction to occur,with mixing of the reaction materials. The sample might be equilibratedwith air or O₂ gas throughout the reaction time period.

The secretome obtained from growth of ActE on cellulose provides allnecessary enzymes for most efficient breakdown of cellulose tocellobiose and mannan to mannose. Weak reaction is observed forbreakdown of xylan to xylose and a mixture of mannobiose and mannose.

The secretome obtained from growth of ActE on xylan provides allnecessary enzymes for most efficient breakdown of xylan to xylobiose andxylose. Weak reaction is observed for breakdown of cellulose tocellobiose and for breakdown of mannan to mannose.

The secretome obtained from growth of ActE on chitin provides allnecessary enzymes for most efficient breakdown of chitin toN-acetylglucosamine. Weak reaction is observed for breakdown of xylan toxylose. Weak reaction is observed for breakdown of cellulose tocellobiose and for breakdown of mannan to mannose.

The secretome obtained from growth of ActE on switchgrass biomassprovides all of the necessary enzymes for breakdown of cellulose, xylan,and mannan contained in switchgrass to the constituent monosaccharidesand disaccharides. Growth of ActE on switchgrass exposed to differentchemical pretreatments changes the composition of enzymes present, whichalters the rate of production and yield of the constituentmonosaccharides and disaccharides.

The secretome obtained from growth of ActE on cellulose provides thenecessary enzymes for breakdown of cellulose to cellobiose. ActE usescellobiose as the growth substrate, so no enzymes are present to convertcellobiose to glucose.

In order to obtain glucose, a cellobiase or beta-glucosidase would beadded. This is a standard practice in biofuels enzymology.

In order to convert cellobiose to glucose, a cellobiase orbeta-glucosidase would be added. Addition of cellulases from otherorganisms can improve the rate of hydrolysis of cellulose, e.g.,addition of CelLcc_CBM3a, an engineered enzyme from C. thermocellumcovered in Fox and Elsen Patent Application No.: PCT/US2010/037094.

The secretome obtained from growth of ActE on cellulose provides all ofthe necessary enzymes for breakdown of cellulose to cellobiose in asoluble form. One skilled in the art might purify these proteinsdirectly from the secretome without use of tags or recombinantapproaches.

As previously noted, the dominance of cellobiose as a product ofcellulose deconstruction by ActE might help to channel cellulolyticactivity to only a subset of the diverse microbes found in the Sirexcommunity. Exploiting this community interaction, along withestablishing control of the highly regulated patterns of gene expressionobserved in ActE provides the basis for a new biotechnological route forlignocellulosic digestion. For example, use of ActE secretomes toproduce cellobiose will restrict the use of cellulose as a fermentationsubstrate to only those organisms capable of cellobiose uptake followedby intracellular conversion to glucose and subsequent glycolytic pathwayintermediates. This might be achieved by coupling ActE enzymes with ayeast fermentation strain engineered to contain a specific cellobiosetransporter and an intracellular cellobiose phosphorylase, leading tothe intracellular production of glucose and glucose-1-phosphate.

ActE secretomes can be mixed with cellulosic biomass to convert it tocellobiose and xylose, as in the biofuels industry. For example, onemight (1) mix the secretome with paper waste to convert it to a mixtureof readily fermentable oligo-, di-, and monosaccharides; (2) mix withanimal feeds to increase the digestibility of the biomass to promoteanimal growth; (3) mix with cotton-based textiles for smoothing or otherrefinements; (4) mix with waste from the shrimp industry to processsolid chitin to soluble constituents; (5) mix with mannan-enrichedmaterials to convert them to mannose and mannobiose. One would also findthe secretome useful for commercial food processing or treatment ofcellulosic bezoar found in the human stomach.

One embodiment of the present invention is an isolation or purifiedpreparation of Streptomyces sp. ActE.

An isolation of ActE was originally reported by Adams et al., (2011)ISME j doi:10.1038/ismej.2011.14, where it was stated that “Sirexnoctilio were collected from infested scots pine, Pinus sylvestris L, inOnondaga County, N.Y., USA in 2008”, and “Microbial isolates wereobtained from four adult females and six larvae collected in 2008, andwere screened for cellulase activity.” These isolates were screened forcellulolytic ability by growing them on CMC, AFEX-treated corn stover,and microcrystalline cellulose.

Applicants envision that one would wish to prepare ActE isolates onspecific nutrient sources for optimization for particular digestionprofiles. Therefore, one may wish to prepare ActE on substrates whereinat least 40%, preferably 85% of Streptomyces sp. ActE's carbon source inthe substrate is derived from a material selected from the groupconsisting of cellulose, cellulose/hemicelluloses mixture,hemicelluloses, xylan, non-wood biomass, wood biomass, and chitin.

In a preferred embodiment, ActE would be grown aerobically to maximizethe secretion of enzymes that include both oxidative and hydrolyticenzymes capable of the rapid deconstruction of biomass. Since ActEcannot utilize mannose for growth, but efficiently liberates mannosefrom biomass, mannose would become available for growth of the inoculumof a fermentation organism in co-culture. The likely fact that ActEproduces at least one antibiotic that would help maintain culturesterility is another possible advantage to establishment of an effectiveco-culture.

The high capacity for mannan hydrolysis coupled with the inability ofActE to use mannose as a growth substrate offers unique potentialopportunity for expansion of deconstruction enzymology to the use ofwoody substrates. The deconstruction of woody substrates is consideredto be more challenging for biofuels production despite the fact thatwoody substrates are also considerably more highly enriched in mannanthan grass substrates. This unique potential opportunity will beenhanced by ongoing plant engineering research efforts to redefine theproportion of xylan and mannan in plant hemicellulose. The availabilityof plant material enriched in mannan will be coupled to vigorousconversion to mannose by ActE secretomes, providing a targeted, simplyfermented C6 sugar for exclusive use by the fermentation organism.

When sufficient titer of enzymes and fermentation organism have beenachieved, facilitated by the vigorous, obligate aerobic growth of ActEand corresponding deconstruction of biomass, the fermentation could beinitiated by removal of the air source from the culture vessel. In theanoxic conditions, ActE would cease to grow, and perhaps even lyse tobecome a protein source for the fermentation organism, which willcontinue to grow on biomass that is simultaneously being deconstructedby the loading of highly active hydrolytic enzymes originally producedby ActE during the aerobic growth phase.

Applicants envision adding an ActE isolate directly to biomass slurry.More preferably an ActE isolate would be added to the pretreatedbiomasses in the enzyme hydrolysis step, because ActE is able to grow atwide range of pH. ActE can be genetically modified so that theproteolysis proof secretome will be achieved. Growth on switchgrasselicits the appearance of new proteins in the secretome that enhance thedegradative ability of the secretome for the plant biomass materials.Applicants envision that the present invention would apply to thebiomasses pretreated by many pretreatment methods comprising AFEX, ionicliquid pretreated, acid hydrolysis, steam explosion, organosolve,sulfite pretreatment to overcome recalcitrance of lignocellulose(SPORL), metal-catalyzed hydrogen peroxide, alkaline wet oxidation andozone pretreatment.

In one preferred embodiment of the present invention, at least one keyenzyme in the secretome can be overexpressed by genetic modification ofthe ActE strain. Table 1 provides various combinations of genes that canbe overexpressed. For example, one may wish to overexpress corecellulose deconstructing enzymes, SACTE_(—)0237 (SEQ ID NOs:1 and 17),SACTE_(—)0482 (SEQ ID NOs:4 and 20), SACTE_(—)0236 (SEQ ID NOs:2 and18), or SACTE_(—)3159 (SEQ ID NOs:3 and 19) together with one or more ofSACTE_(—)2347 (SEQ ID NOs:6 and 22), and SACTE_(—)0265 (SEQ ID NOs:5 and21). One may wish to overexpress core xylan deconstructing enzymes,SACTE_(—)0265 (SEQ ID NOs:5 and 21), SACTE_(—)0358 (SEQ ID NOs:8 and24), SACTE_(—)0357 (SEQ ID NOs:7 and 23), SACTE_(—)5978 (SEQ ID NOs:16and 32), and SACTE_(—)5230 (SEQ ID NOs:33 and 48). One may wish tooverexpress core mannan deconstruction enzymes, such as SACTE_(—)2347(SEQ ID NOs:6 and 22). Additionally, SACTE_(—)4755 (SEQ ID NOs:13 and29) and SACTE_(—)4738 (SEQ ID NOs:12 and 28) may be overexpressed forbeta-1,3-glucan deconstruction. One may also overexpress all or some ofthe aforementioned genes for efficient biomass deconstruction.

In another embodiment of the present invention, at least one key enzymein the secretome can be overexpressed and secreted by geneticmodification of a different microbial host such as Streptomyceslividans, which is used for industrial secretion of proteins (Anne andVan Mellaert. (1993)), or T. reesei, which is used for secretion ofenzymes in the biofuels industry (Saloheimo and Pakula, Microbiology,Epub date 2011 Nov. 5).

In another embodiment of the present invention, at least one key enzymein the secretome can be overexpressed by genetic modification of adifferent microbial host such as S. cerevisiae or E. coli such that theexpressed protein will be retained inside of the host cell. The hostcells would then be harvested and used as a delivery agent without needfor purification of the entrained enzyme, as described in Wood et al.,1997. This version of the invention may be useful in the enzymaticpretreatment of agricultural crop materials for consumption by ruminantanimals.

Combinations of ActE Genes and Expression Products

Selected minimal genes in each subset were chosen based on thecombination of genomic, transcriptomic and secretomic results (SeeExamples and Table 1). For example, in the cellulose minimal gene set,expression of these genes was relatively enriched in cellulose growncells, compared to glucose grown cells, also corresponding proteins werehighly secreted in response to the cellulose in culture medium. Selectedminimal genes were annotated to have cellulose utilization function. Alarger set of genes for cellulose utilization were selected based on theenrichment of gene expression in cellulose-grown cells relative toglucose-grown cells, and a functional annotation supports celluloseutilization of these genes. Additionally, neighborhood genes to theseselected genes on genome were included as genes regulated under samepromoter. Similarly, both minimal and a large set of genes for xylan,chitin, and biomasses were elected.

In one embodiment, the present invention is a composition useful fordigesting lignocellulosic material comprising genes or expressionproducts thereof selected from the group consisting of: (a)SActE_(—)0237 (SEQ ID NOs:1 and 17), SActE_(—)0236 (SEQ ID NOs:2 and18), SActE_(—)3159 (SEQ ID NOs:3 and 19), SActE_(—)0482 (SEQ ID NOs:4and 20), SActE_(—)0265 (SEQ ID NOs:5 and 21), and SActE_(—)2347 (SEQ IDNOs:6 and 22), and (b) SActE_(—)0357 (CE4) (SEQ ID NOs:7 and 23),SActE_(—)0358 (GH11) (SEQ ID NOs:8 and 24), SActE_(—)1310 (PL3) (SEQ IDNOs:9 and 25), SActE_(—)3717 (GH9) (SEQ ID NOs:10 and 26), SActE_(—)4638(SEQ ID NOs:11 and 27), SActE_(—)4738 (GH16) (SEQ ID NOs:12 and 28),SActE_(—)4755 (GH64) (SEQ ID NOs:13 and 29), SActE_(—)5457 (GH46) (SEQID NOs:14 and 30), SActE_(—)5647 (GH87) (SEQ ID NOs:15 and 31), andSActE_(—)5978 (PL1) (SEQ ID NOs:16 and 32). In a preferred embodiment,the composition comprises at least three or four of the genes orexpression products.

In one embodiment, one would use at least one member of (a) to digest apreferred lignocellulosic material.

In another embodiment, one would use at least the first four members[SActE_(—)0237 (SEQ ID NOs:1 and 17), SActE_(—)0236 (SEQ ID NOs:2 and18), SActE_(—)3159 (SEQ ID NOs:3 and 19), and SActE_(—)0482 (SEQ IDNOs:4 and 20)] of (a) to digest a preferred lignocellulosic material.

In another embodiment, one would use at least one member of (a) and atleast one member from (b), to digest a preferred lignocellulosicmaterial.

In a preferred embodiment, one would use all the members of (a) and (b),to digest a preferred lignocellulosic material.

In other embodiments, the combination of genes or expression productsthereof in the present invention is dependent on the specificlignocellulosic material to be digested. In one embodiment, acomposition optimized for cellulose utilization may include anycombinations of ActE genes and expression products disclosed above withat least one member selected from SActE_(—)0265 (GH10) (SEQ ID NOs:5 and21) and SActE_(—)2347 (GH5) (SEQ ID NOs:6 and 22) genes or expressionproducts thereof.

In another embodiment, a composition optimized for xylan utilization mayinclude any combinations of ActE genes and expression products disclosedabove with at least one member selected from SActE_(—)0265 (GH10) (SEQID NOs:5 and 21), SActE_(—)0358 (GH11) (SEQ ID NOs:8 and 24),SActE_(—)0357 (CE4) (SEQ ID NOs:7 and 23), SActE_(—)5978 (PL1) (SEQ IDNOs:16 and 32) and SActE_(—)5230 (xylose isomerase) (SEQ ID NOs:33 and48) genes or expression products thereof. In a preferred embodiment, thecomposition comprises at least three or four of the genes or expressionproducts.

In another embodiment, a composition optimized for chitin utilizationmay include any combinations of ActE genes and expression productsdisclosed above with at least one member selected from SActE_(—)4571(GH18) (SEQ ID NOs:34 and 49), SActE_(—)2313 (CBM33) (SEQ ID NOs:35 and50), SActE_(—)4246 (GH18) (SEQ ID NOs:36 and 51), SActE_(—)3064 (GH19)(SEQ ID NOs:37 and 52), and SActE_(—)5764 (GH18) (SEQ ID NOs:38 and 53)genes or expression products thereof. In a preferred embodiment, thecomposition comprises at least three or four of the genes or expressionproducts.

In another embodiment, a composition optimized for biomass utilizationmay include any combinations of ActE genes and expression productsdisclosed above with SActE_(—)5457 (GH46) (SEQ ID NOs:14 and 30) genesor expression products thereof.

In another embodiment, a composition optimized for mannan utilizationmay include any combinations of ActE genes and expression productsdisclosed above with SactE_(—)2347 (GH5) (SEQ ID NO:6 and 22) genes orexpression products thereof.

In another embodiment, a composition optimized for beta-1,3-glucanutilization may include any combinations of ActE genes and expressionproducts disclosed above with at least one member selected fromSActE_(—)4755 (GH64) (SEQ ID NOs:13 and 29) and SActE_(—)4738 (GH16)(SEQ ID NOs:12 and 28) genes or expression products thereof.

In another embodiment, a composition optimized for pectin releaseutilization may include any combinations of ActE genes and expressionproducts disclosed above with SActE_(—)1310 (PL3) (SEQ ID NOs:9 and 25)gene or expression products derived thereof.

In another embodiment, a composition optimized for alginate releaseutilization may include any combinations of ActE genes and expressionproducts disclosed above with SActE_(—)4638 (SEQ ID NOs:11 and 27) geneor expression products derived thereof.

In another embodiment, a composition optimized for galactose releaseutilization may include any combinations of ActE genes and expressionproducts disclosed above with SactE_(—)5647 (GH87) (SEQ ID NOs:15 and31) gene or expression products derived thereof.

In another embodiment, the present invention is summarized as acomposition useful for xylan degradation comprising SActE_(—)0265 (GH10)(SEQ ID NOs:5 and 21) and SActE_(—)0358 (GH11) (SEQ ID NOs:8 and 24)genes or expression products thereof.

In another embodiment, the present invention is summarized as acomposition useful for xylan degradation comprising SActE_(—)0265 (GH10)(SEQ ID NOs:5 and 21), SActE_(—)0358 (GH11) (SEQ ID NOs:8 and 24),SActE_(—)0265 (GH10) (SEQ ID NOs:5 and 21), SActE_(—)0358 (GH11) (SEQ IDNOs:8 and 24), SActE_(—)0357 (CE4) (SEQ ID NOs:7 and 23), SActE_(—)5978(PL1) (SEQ ID NOs:16 and 32), and SActE_(—)5230 (xylose isomerase) (SEQID NOs:33 and 48) genes or expression products thereof. In a preferredembodiment, the composition comprises at least three or four of thegenes or expression products.

In another embodiment, the present invention is summarized as acomposition useful for biomass degradation comprising SActE_(—)0237(GH6) (SEQ ID NOs:1 and 17), SActE_(—)0482 (GH5) (SEQ ID NOs:4 and 20),SActE_(—)3159 (CBM33) (SEQ ID NOs:3 and 19), SActE_(—)0236 (GH48) (SEQID NOs:2 and 18), SActE_(—)3717 (GH9) (SEQ ID NOs:10 and 26),SActE_(—)0265 (GH10) (SEQ ID NOs:5 and 21), SActE_(—)0358 (GH11) (SEQ IDNOs:8 and 24), SActE_(—)2347 (GH5) (SEQ ID NOs:6 and 22) andSActE_(—)1310 (PL3) (SEQ ID NOs:9 and 25) genes or expression productsthereof. In a preferred embodiment, the composition comprises at leastthree or four of the genes or expression products.

In one embodiment, the present invention is a composition useful fordigesting lignocellulosic material comprising genes or expressionproducts thereof selected from the group consisting of: (a)SActE_(—)0237 (SEQ ID NOs:1 and 17), SActE_(—)0236 (SEQ ID NOs:2 and18), SActE_(—)3159 (SEQ ID NOs:3 and 19), SActE_(—)0482 (SEQ ID NOs:4and 20), SActE_(—)0265 (SEQ ID NOs:5 and 21), and SActE_(—)2347 (SEQ IDNOs:6 and 22) (for cellulose); (b) SActE_(—)0265 (SEQ ID NOs:5 and 21),SActE_(—)0357 (SEQ ID NOs:7 and 23), SActE_(—)0358 (SEQ ID NOs:8 and24), SActE_(—)5230 (SEQ ID NOs:33 and 48) and SActE_(—)5978 (SEQ IDNOs:16 and 32) (for xylan); (c) SActE_(—)2313 (SEQ ID NOs:35 and 50),SActE_(—)3064 (SEQ ID NOs:37 and 52), SActE_(—)4246 (SEQ ID NOs:36 and51), SActE_(—)4571 (SEQ ID NOs:34 and 49) and SActE_(—)5764 (SEQ IDNOs:38 and 53) (for chitin); (d) SActE_(—)2347 (SEQ ID NOs:6 and 22)(for mannan); and (e) SActE_(—)0236 (SEQ ID NOs:2 and 18), SActE_(—)0237(SEQ ID NOs:1 and 17), SActE_(—)0265 (SEQ ID NOs:5 and 21),SActE_(—)0358 (SEQ ID NOs:8 and 24), SActE_(—)1310 (SEQ ID NOs:9 and25), SActE_(—)2347 (SEQ ID NOs:6 and 22) and SActE_(—)3159 (SEQ ID NOs:3and 19) (for biomass). In a preferred embodiment, the compositioncomprises at least three or four of the genes or expression products.

In one embodiment, one would use at least two members of (a), (b), (c),(d) or (e) to digest a preferred lignocellulosic material.

In another embodiment, one would use at least three members.

In a preferred embodiment, one would use all members of (a), (b), (c),(d) or (e).

In another embodiment, one would add gene expression products from thelist in Table 1 to a substrate to be digested. For example, forpreferred cellulose digestion, one would select at least two members of(a), as described above, and at least one member of the “additionaluseful genes” in Table 1.

In the case of cellulose degradation, the inventors believeSACTE_(—)3159 (SEQ ID NOs:3 and 19), SACTE_(—)0237 (SEQ ID NOs:1 and17), SACTE_(—)0482 (SEQ ID NOs:4 and 20), and SACTE_(—)0236 (SEQ IDNOs:2 and 18) act cooperatively to create nicks and hydrolyze cellobioseunits from crystalline cellulose.

ActE key genes can be transferred into known cellulolytic organisms inorder to enhance the cellulolytic ability of these organisms. Acellulolytic fungus, T. reesei, has been studied for industrialapplications, and can be genetically modified. Applicants' data supportsynergism of cellulolytic ability of enzymes from different species. Achromosomal gene transfer can be performed into T. reesei by protoplasttransformation with a high copy plasmid carrying one or more of the ActEcellulolytic key genes.

A chromosomal or a non-chromosomal gene transfer can be made into ayeast species such as Saccharomyces cerevisiae. For non-chromosomal genetransfer, a high copy plasmid carrying a cassette of five minimal genes(SACTE_(—)0236 (SEQ ID NOs:2 and 18), SACTE_(—)0237 (SEQ ID NOs:1 and17), SACTE_(—)0482 (SEQ ID NOs:4 and 20), SACTE_(—)3717 (SEQ ID NOs:10and 26) and SACTE_(—)3159 (SEQ ID NOs:3 and 19)) would be used to confercellulolytic and mannanolytic capability to the yeast strain. Similarapproaches could be used to confer xylanolytic and chitinolyticcapability using combinations of the genes described herein.

One might wish to recombinantly express the disclosed enzymes in E. coliin order to achieve high yield of each enzyme. As is shown in thesynergistic result in Example 18, cellulose degradation can be improvedby combination of ActE enzymes to enzymes from other organisms.

FIG. 18 shows Spectra count of proteins identified on each substrate,where top 95% most abundant proteins were highlighted green, lightpurple, purple, blue, orange, pink, light blue and yellow on glucose,cellobiose, cellulose, xylan, switchgrass, AFEX-SG, IL-SG and chitin,respectively.

Applicants envision that one would use a composition comprising at leastone member of the abundant proteins, e.g., those highlighted proteins inFIG. 18, for digesting the corresponding lignocellulosic materials. Forexample, to digest a cellulose material, one would choose at least onegene or expression products thereof selected from the group consistingof SACTE_(—)0237 (SEQ ID NOs:1 and 17), SACTE_(—)0236 (SEQ ID NOs:2 and18), SACTE_(—)2347 (SEQ ID NOs:6 and 22), SACTE_(—)3159 (SEQ ID NOs:3and 19), SACTE_(—)0482 (SEQ ID NOs:4 and 20), SACTE_(—)0265 (SEQ IDNOs:5 and 21), SACTE_(—)0357 (SEQ ID NOs:7 and 23), SACTE_(—)4439 (SEQID NOs:39 and 54), SACTE_(—)0562 (SEQ ID NOs:40 and 55), SACTE_(—)0358(SEQ ID NOs:8 and 24), SACTE_(—)4343 (SEQ ID NOs:41 and 56),SACTE_(—)1546 (SEQ ID NOs:42 and 57), SACTE_(—)1310 (SEQ ID NOs:9 and25), SACTE_(—)4638 (SEQ ID NOs:11 and 27), SACTE_(—)5668 (SEQ ID NOs:45and 60), SACTE_(—)3717 (SEQ ID NOs:10 and 26), SACTE_(—)3590 (SEQ IDNOs:43 and 58), SACTE_(—)2172 (SEQ ID NOs:44 and 59), SACTE_(—)4571 (SEQID NOs:34 and 49), SACTE_(—)5978 (SEQ ID NOs:16 and 32), SACTE_(—)6428(SEQ ID NOs:46 and 61), SACTE_(—)2313 (SEQ ID NOs:35 and 50), andSACTE_(—)0366 (SEQ ID NOs:47 and 62). In a preferred embodiment, thecomposition comprises at least three or four of the genes or expressionproducts.

In one preferred embodiment, one would use all the highlighted proteinsfor digesting the corresponding lignocellulosic materials.

In another embodiment, one would add gene expression products from thelist in Table 1 to a substrate to be digested. For example, forpreferred cellulose digestion, one would select at least one member ofthe abundant proteins, as described above, and at least one member ofthe “additional useful genes” in Table 1.

TABLE 1 ActE genes or expression products useful for lignocellulosicdegradation. Gene or Expression Product Combinations Preferred subsetsAdditional Useful Genes SACTE_0236, SACTE_0237, Cellulose SACTE_0229,SACTE_0230, SACTE_3159, SACTE_0482 degradation SACTE_0231, SACTE_0232,and SACTE_3717 SACTE_0233, SACTE_0234, SACTE_0235, SACTE_0480,SACTE_0481, SACTE_0483, SACTE_0562, SACTE_0563, SACTE_0733, SACTE_0734,SACTE_2286, SACTE_2287, SACTE_2288, SACTE_2289, SACTE_3158, SACTE_4737,and SACTE_6428 SACTE_0265, SACTE_0357, Xylan degradation SACTE_0364,SACTE_0365, SACTE_0358, SACTE_5230 SACTE_0366, SACTE_0368, andSACTE_5978 SACTE_0369, SACTE_0370, SACTE_0527, SACTE_0528, SACTE_5227,SACTE_5228, SACTE_5229, SACTE_5858, and SACTE_5859 SACTE_2313,SACTE_3064, Chitin degradation SACTE_0080, SACTE_0081, SACTE_4246,SACTE_4571 SACTE_0844, SACTE_0846, and SACTE_5764 SACTE_0860,SACTE_3063, SACTE_4858, SACTE_6493 and SACTE_6494 SACTE_2347 Mannandegradation SACTE_1310 Pectin degradation SACTE_4638 Alginate releaseSACTE_5647 Galactose release SACTE_5648 SACTE_4738 and Beta-1,3-glucanSACTE_4737, SACTE_4739 and SACTE_4755 degradation SACTE_4756 SACTE_0236,SACTE_0237, Cellulose and SACTE_3065, SACTE_4730, SACTE_0265,SACTE_0358, hemicelluloses SACTE_4755, and SACTE_5166 SACTE_0482,SACTE_1310, degradation SACTE_2347, SACTE_3159 and SACTE_3717

In one embodiment, the present invention is a method for digesting alignocellulosic material, comprising exposing the material to asufficient amount of a composition of enzymes, wherein the exposedmaterial is at least partially digested. The enzymes may be ActEsecretomes, and ActE secretomes may be prepared and isolated using themethods described above.

In another embodiment, the composition of enzymes for a method fordigesting a lignocellulosic material may include ActE secretomes in acombination with secretomes from other organisms, or with enzymes orenzyme compositions, such as Spezyme CP, to increase the activity ofboth preparations by synergy of the enzymes contained in eachpreparation.

In another embodiment, the composition of enzymes for a method fordigesting a lignocellulosic material may be any combinations of ActEgenes and expression products as described above.

EXAMPLES

Materials and Methods

Genome Analysis.

The complete genome sequence of Streptomyces sp. SirexAA-E (ActE,taxonomy ID 862751) was determined by the Joint Genome Institute,project ID 4086644. Gene annotation models were predicted using Prodigal(Hyatt, et al., 2010), examined using Artemis (Rutherford, et al.,2000), and are available at NCBI with the following accession numbers,GenBank: CP002993.1; RefSeq: NC_(—)015953.1. Carbohydrate-active enzymeswere annotated by comparison of all translated open-reading frames tothe CAZy database (Cantarel, et al., 2009). We collected CAZy annotatedgenes from the CAZy database (www.cazy.org). We then used BLASTP tocompare all ActE protein-coding sequences to the CAZy database and tothe pfam database(ftp://ftp.ncbi.nih.gov/pub/mmdb/cdd/little_endian/Pfam_LE.tar.gz).These two annotations were then crosschecked, and proteins annotated byboth databases were identified as our final CAZy annotation. Secretedproteins were identified by SignalP, TatP, and SecretomeP analyses.BLAST was used to identify sequence orthologs in other organisms.Secondary metabolite gene clusters were identified by AntiSmash analysis(Medema, et al., 2011). CebR boxes were identified by using BLASTcomparison of the S. griseus CebR box sequence to the ActE genome(Marushima, Ohnishi, et al., 2009). Networks of expression andfunctional categories were visualized using Cytoscape (Shannon, et al.,2003)

Biomass Substrates.

Switchgrass and AFEX-treated switchgrass were obtained from Great LakesBioenergy Research Center. Extensively washed ionic liquid-treatedswitchgrass was the generous gift of Dr. Masood Hadi (Joint BioEnergyInstitute). Wood kraft pulp preparations were the generous gift of Dr.Xuejun Pan (University of Wisconsin Department of BiosystemsEngineering).

Growth of Organisms.

ActE, S. coelicolor, S. griseus and T. reesei RUT-C30 were grown at pH6.0 and ActE was also grown at pH 6.9 in M63 minimal medium, where 1 Lcontains: 10.72 g K₂HPO₄; 5.24 g KH₂PO₄; 2 g (NH₄)₂SO₄; 0.5 mL ironsulfate (1 mg/mL in 0.01 M HCl); 1 mL 1 M MgSO₄; 1 mL thiamine solution(1 mg/mL) supplemented with glucose, cellulose (either Whatman #1 filterpaper or Sigmacell-20, Sigma/Aldrich, St. Louis, Mo. as indicated),xylan, chitin, switchgrass, AFEX-treated switchgrass (Balan et al.,2009), or ionic liquid-treated switchgrass as the sole carbon source(0.5% w/v). Cultures were incubated for 7 days at 30° C. with shaking.In this medium at pH 6.9, ActE has doubling times of 2.5 h for growth onxylan and switchgrass, 8 h for glucose and 13 h for cellulose asdetermined by time-dependent increases in total protein present in theculture medium.

RNA Microarray.

ActE was grown in minimal medium plus the indicated substrate for 7days. The cell pellet was separated from the culture medium bycentrifugation for 10 min at 3000×g. Microarray experiments were carriedout as reported previously (Riederer, et al., 2011). The total RNA wasextracted from the cell pellet and purified. The University of WisconsinGene Expression Center carried out the syntheses of cDNA and arrayhybridizations. Four-plex arrays were constructed by Nimblegen andhybridized with 10 μg of labeled cDNA. ArrayStar (v4.02, DNASTAR,Madison, Wis.) was used to quantify and visualize data. All analyseswere based on three or more biological replicates per carbon source.Quantile normalization and robust multi-array averaging (RMA) wereapplied to the entire data set. Unless otherwise specified, expressionlevels are based on log 2 values and statistical analysis of thedatasets were performed using the moderated t-test.

Preparation of Secretomes.

Supernatants obtained from different culture media were prepared bycentrifugation of the culture medium for 10 min at 3000×g, which removedthe remaining insoluble polysaccharide and adhered cells. Thesupernatant fraction was then passed through a 0.22-μm filter in orderto remove any remaining cells. For enzymatic assays, the secretomes wereconcentrated using a 3-kDa cut off ultrafiltration membrane. Theconcentration of secretome protein was determined by Bradford assay, andthe typical yield was ˜150-300 mg of total secreted protein per liter ofculture medium.

Extracellular Protein Profiles.

Extracellular proteins from culture secretomes were precipitated withtrichloroacetic acid (TCA), resuspended in denaturing sample buffer (SDSand 2-mercaptoethanol), and separated by SDS-PAGE in 4-20% gels. Proteinbands of interest were excised from the gel, digested with trypsin,desalted with C18 pipette tips (Millipore, Billerica, Mass.) andidentified by MALDI-TOF (MDS SCIEX 4800 MALDI TOF/TOF, AppliedBiosystems, Foster City, Calif.). Additional samples from the sameculture secretomes were analyzed by LC-MS/MS to identify highly abundantproteins in the sample.

Ion Exchange Separation of the ActE Secretome.

The ActE cellulose secretome was diluted with cold deionized water untilthe ionic strength was less than 50 mS. The diluted sample was loadedonto an AKTApürifier™ chromatography station equipped with a 16/10 MonoQFF ion exchange column. The column was washed with 100 mL of 10 mMphosphate, pH 6.0, to remove unbound proteins. The bound proteins wereeluted in a linear, 200 mL gradient of NaCl from 0 to 0.8 M in the samebuffer. Fractions from the gradient elution were collected and separatedby SDS PAGE. The proportional contribution of individual proteins ineach fraction was estimated from SDS PAGE. Individual protein bands fromeach fraction were cut from the gel and submitted for LC-MS/MS analysisto confirm their identities.

LC-MS/MS Analyses.

These experiments were performed at the University of WisconsinBiotechnology Center. Samples were prepared by TCA precipitation of 100ng of total secreted protein from 7-day old culture supernatants.Protein samples were digested with trypsin (sequencing grade trypsin,Promega, Madison, Wis.) and were desalted using C18 pipette tips(Millipore, Billerica, Mass.). High-energy collision dissociation (HCD)MS analyses employing a capillary LC-MS/MS were performed on anelectrospray ionization FT/ion-trap mass spectrometer (LTQ Orbitrap XL,Thermo Fisher Scientific, San Jose, Calif.). The MS and MS/MS spectrawere searched against the spectra obtained from the ActE proteome byusing Scaffold (Scaffold_(—)3_(—)00_(—)06, Proteome Software, Portland,Oreg.).

Enzyme Activity Measurements.

Reducing sugar assays were carried out by mixing secretome preparationswith polysaccharide-containing substrates including cellulose (eitherWhatman #1 filter paper or Sigmacell-20 as indicated), xylan, chitin,mannan, switchgrass, AFEX pretreated switchgrass, or ionic-liquidpretreated switchgrass²⁴. After incubation in 0.1 M sodium phosphate, pH6 at 40° C. for 20 h, the reducing sugar content was detected bydinitrosalicylic acid assay (Miller, 1959) and calibrated by usingglucose, xylose, or mannose as standards. Purified polysaccharidepreparations had negligible background response in the absence of addedenzymes. Cellobionic and gluconic acids were assayed by a coupled enzymeassay (K-GATE system, Megazyme, Bray Ireland). SPEZYME CP was obtainedfrom Genencor with batch number #4901522860. The distributions ofsoluble sugar oligomers obtained from secretome reactions weredetermined using a Shimadzu Liquid Chromatograph HPLC system (ShimadzuScientific Instruments, Columbia, Md.) equipped with a refractive indexdetector (RID-10A) and a Phenomenex Rezex RPM-monosaccharide column. Thetemperature was maintained at 85° C. and Milli-Q water was used as themobile phase at 0.6 mL min⁻¹ flow rate. Glucose, cellobiose,cellotriose, cellotetraose, cellopentaose, and cellohexaose (Sigma) wereused as standards. The integrated areas of peaks were analyzed by EZstart 7.2 SP1 software (Shimadzu).

Fractions obtained from the ion exchange separation of the ActEcellulose secretome were combined as unary, binary, ternary, andquaternary assemblies where the total protein concentration was fixedand the individual fractions contributed all, halves, thirds, orquarters of the total protein. The most active fraction was assembledfrom a ternary combination of fractions containing the followingenzymes: fraction 1, SACTE_(—)3159 (CBM33/CBM2 oxidative endocellulase,95%) and SACTE_(—)4738 (GH16 β-1,3 endoglucanase, 5%); fraction 2,SACTE_(—)0237 (GH6 exocellulase, 60%), SACTE_(—)0482 (GH5 endocellulase,25%), SACTE_(—)0237 (β-1,3 glucanase, 10%) and SACTE_(—)3159 (oxidativeendocellulase, <5%); and fraction 3, SACTE_(—)0236 (GH48 exocellulase,75%), SACTE_(—)3717 (GH9 endocellulase, 20%) and SACTE_(—)5457 (GH46chitinase, 5%).

Cellobionic and gluconic acids were assayed by a coupled enzyme assay(K-GATE system, Megazyme, Bray Ireland), either with or without theaddition of a large excess of β-glucosidase (Cat. No. 31571, Lucigen,Middleton, Wis.).

Two lots of Spezyme CP were obtained from Genencor (#4900901244, Jan.27, 2010 and #4901522860, Sep. 2, 2011). The specific activity of thesetwo preparations was indistinguishable.

HPLC Analysis.

The distributions of soluble sugar oligomers obtained from secretomereactions without and with the addition of excess β-glucosidase(Lucigen) were determined using a Shimadzu Liquid Chromatograph HPLCsystem (Shimadzu Scientific Instruments, Columbia, Md.) equipped with arefractive index detector (RID-10A) and a Phenomenex RezexRPM-monosaccharide column. The temperature was maintained at 85° C. andmilli-Q water was used as the mobile phase at 0.6 mL min⁻¹ flow rate.Glucose, cellobiose, cellotriose, cellotetraose, and cellopentaose(Sigma) were used as standards. The integrated areas of peaks wereanalyzed by EZ start 7.2 SP1 software (Shimadzu).

For the experiments shown in FIG. 21, the ActE secretome (1 μg totalprotein); CelLcc_CBM3a (1 μg); ActE secretome (0.5 μg) and CelLcc_CBM3a(0.5 μg); or Spezyme CP (1 μg total protein) were used. The products ofthe enzyme reactions detected by HPLC were: ActE secretome, 95%cellobiose, 5% glucose; CelLcc_CBM3a reaction, 90% cellobiose, 10%glucose; ActE & CelLcc_CBM3a, 5% cellotetraose, 80% cellotriose, 15%cellobiose; Spezyme CP, 33% cellobiose, 67% glucose. All products couldbe converted to glucose in the presence of excess β-glucosidase.

CelLcc_CBM3a. The nucleotide and amino acid sequence of CelLcc_CBM3a isshown in FIG. 22. CelRcc_CBM3a is an engineered exoglucanase composed ofthe catalytic core of C. thermocellum CelL (Cthe_(—)0405, residues 32 to429) fused to a C. thermocellum-derived linker sequence and the CBM3adomain from Cthe_(—)3077, the CipA scaffoldin. This construct wascreated to better understand the performance of enzymes that arenormally targeted to the clostridial cellulosome. The replacement of thedockerin domain in Cthe_(—)0405 with the CBM3a domain abrogates the needfor a cellulosomal attachment to obtain maximal catalytic activity fromCelLcc_CBM3a on solid substrates. The indicated nucleotide sequence wassub-cloned into wheat germ cell-free translation (Makino et al., 2010)and E. coli expression vectors (Blommel et al., 2009) for proteinproduction. CelLcc_CBM3a was purified by standard immobilized metal(Ni²⁺) chromatography. There was no difference in the specific activityof the protein prepared by these two methods.

Example 1 ActE Exhibits High Cellulolytic Activity Relative to OtherCellulolytic Organisms

Prokaryotes such as Streptomyces are often easier to grow thaneukaryotes (i.e., fungi such as T. reesei), and aerobes are often easierand more energetically efficient to grow than anaerobes. Streptomycesmay also have an advantage of producing antibiotics that limit theability of other organisms to contaminate the culture medium duringgrowth (Galm et al., 2011; Susi et al., 2011). This may be of advantageduring large-scale culture with non-sterile biomass materials such aswill be encountered in the biofuels industry.

When compared to other cellulolytic organisms (FIG. 1 and FIG. 6), ActEgrows well on pure cellulose substances including amorphous cellulose(cellulose treated with phosphoric acid so as to remove all crystallinestructure), filter paper (containing a mixture of amorphous andcrystalline cellulose) and Sigmacell (primarily in the crystalline stateas determined by X-ray powder diffraction), as well as otherpolysaccharides such as beta-1,3-glucan (callose), xylan, and chitin.ActE also grows well on biomass samples such as corn stover,ammonia-fiber expansion pretreated corn stover, switchgrass,ammonia-fiber expansion pretreated switchgrass, ionic liquids pretreatedswitchgrass, bleached spruce wood kraft pulp, and unbleached lodgepolepine kraft pulp.

FIG. 1 compares the ability of ActE, S. coelicolor A3(2) (NCBI taxonomyID 100226) and S. griseus (NCBI CP002993.1; RefSeq: NC_(—)015953.1) togrow in minimal medium containing filter paper as the only carbon andenergy source. These images demonstrate the considerably differentcapabilities of the three ostensibly cellulolytic organisms. Thus ActEcompletely destroys the filter paper and achieves high cell density,while the two other, reputedly highly cellulolytic strains are onlycapable of weak colony formation attached to the filter paper. Thisresult establishes that ActE has uniquely high cellulolytic capacityrelative to other Streptomyces strains reported to also have thiscapability (Forsberg et al., 2011). In fact, the images of FIG. 1 andFIG. 6 demonstrate ActE has cellulolytic capacity rivaling that of T.reesei strain Rut-C30, which is widely acknowledged to be the industrialbenchmark for cellulolytic capacity (Merino and Chemy, Adv. Biochem.Eng. Biotechnol. 108:95-120, 2007).

Example 2 Pretreatments Useful for Generating Fermentable Sugars

In the biofuels arena, the desired cellulose fractions of plant biomassare protected by the crystalline packing of the individual cellulosestrands, and by the surrounding coating of hemicellulose and lignin. Inorder to most efficiently access the cellulose, chemical pretreatmentsare required to “loosen up” the plant cell wall structure. In thiscontext, “loosen up” may mean removal of the lignin fraction, partialhydrolysis of feruloyl and acetyl esters present in hemicellulose, andchanges in the crystallinity of the cellulose. An optimal pretreatmentretains all fractions of biomass lignin, hemicellulose and cellulose) inphysical states that can be subsequently used by microbes and enzymes assubstrates.

Ammonia-fiber expansion is a pretreatment that uses a combination ofammonia gas, low pressure, and low temperature to effect the looseningprocess (Balan et al., 2009; Chundawat et al., 2011; InternationalPatent Publication No.: WO 2010/125679). It is particularly effectivewith grasses, and retains all fractions of the biomass for subsequentvalorization without introducing water or salts into the biomass. Ionicliquids pretreatment comprises mixing a charged chemical substance(i.e., the ionic liquid) in equal mass proportions with the biomassmaterial. Interactions between the ionic liquid substance and thebiomass cause the crystalline structure of cellulose to convert to anamorphous state (Cheng et al., 2011; Li et al., 2011) but the biomassalso becomes heavily contaminated with the ionic liquid during thispretreatment, requiring extensive washing with water, a valuableresource in many localities. Kraft pulping is a method for production ofpaper from wood that involves treatment of the biomass material withstrong alkali, sodium sulfite and moderate temperature, resulting indestruction of the lignin and hemicellulose from the desired cellulosefraction; the final biomass material is also heavily contaminated withsalts that also requires extensive washing with water to remove. Acidpretreatments retain the lignin and cellulose but destroy thehemicellulose fraction, and in doing so create toxic substances derivedfrom the decomposition of hemicellulose. Because of the need toneutralize the acid, this pretreatment generates a large contaminationof salt that also requires extensive washing with water. SPORL is anacidic pretreatment that uses sulfuric acid, elevated temperature, andsodium bisulfite to effect the pretreatment (Wang et al., 2009; Tian etal., 2011). In SPORL, the lignin and hemicellulose are destroyed andcellulose is recovered, but the cellulose is again heavily contaminatedwith salts and toxic substances derived from chemical decomposition ofhemicellulose.

ActE secretomes are highly effective for degradation of lignocellulosicmaterial pre-treated with AFEX. ActE secretomes are also effective fordegrading lignocellulosic material pretreated with ionic liquids, Kraftpulping, acid or SPORL and for degrading untreated lignocellulosicmaterial.

Example 3 ActE Genome has High Content of Genes Encoding CarbohydrateActive enZymes (CAZy) Relative to Other Cellulolytic Organisms

Protein-coding sequences of the ActE genome (Hyatt et al., 2010) wereanalyzed by BLAST comparison (Altschul et al., 1990) to the CarbohydrateActive enZyme (CAZy) database (Cantarel et al., 2009).

Table 2 compares the genomic characteristics of ActE with well-knownsoil-isolated Streptomyces that produce antibiotics and with two modelcellulolytic bacteria, Clostridium thermocellum and Cellvibrio japonicas(Lynd, Weimer, et al., 2002; Deboy, et al., 2008; Riederer, et al.,2011). Putative biomass-degrading protein-coding sequences from ActEwere identified by BLAST analysis of the finished genome to theCarbohydrate Active enZyme (CAZy) database. Among the 6357 predictedprotein-coding genes, 167 have one or more domains assigned to CAZyfamilies, including 119 glycoside hydrolases (GHs), 29 carbohydrateesterases (CEs), 6 polysaccharide lyases (PLs) and 85 carbohydratebinding modules (CBMs). ActE contains 45 different types of GH families,4 PL families, 7 CE families, and 21 CBM families. The number of totalCAZy domains and diversity of CAZy families is comparable to otherhighly cellulolytic organisms.

TABLE 2 Comparison of genomic composition. ActE S. coelicolor S. griseusC. thermocellum C. japonicus Genome size 7414440 8667507 8545929 38433014576573 (nt) Proteome size 6357 8153 7136 3173 3750 Total CAZy 167 221132 103 183 Proteins % CAZy 2.6% 2.7% 1.8% 3.2% 4.9% Proteins^(a) TotalGH^(b) 119 154 80 70 124 Total PL^(c) 6 11 4 6 14 Total CE^(d) 29 36 2320 28 Total CBM^(e) 85 98 68 121 134 antiSMASH 22 24 37 3 4 clusters^(f)Genes in 620 718 1139 89 111 clusters % antiSMASH 9.8% 8.8% 16.0% 2.8%3.0% ^(a)Proteins classified as Carbohydrate Active Enzymes (CAZy).^(b)GH, glycoside hydrolase. ^(c)PL, pectate lyase. ^(d)CE, carbohydrateesterase. ^(e)CBM, carbohydrate binding module. ^(f)Putative antibioticproducing gene cluster.

Nearly all publically available Streptomyces genomes encode a relativelyhigh percentage of genes for putative cellulolytic enzymes.Interestingly, ActE and the antibiotic producing Streptomyces, S.griseus and S. coelicolor, shown in Table 2 have similar numbers andcompositions of CAZy families, but substantially different genome sizes.However, these antibiotic-producing Streptomyces are not highlycellulolytic (FIG. 1). Relative to S. griseus and S. coelicolor, theActE genome contains two unique CAZy families but does not possess 16CAZy families present in these species. However, ActE contains morerepresentatives in 13 CAZy families. Enrichment of certain CAZy familieswas observed in other highly cellulolytic organisms. For example, C.thermocellum contains 16 genes in the GH9 family alone. It isinteresting to consider whether the reduction in total genome size anddifferences in CAZy composition between ActE and other closely relatedsoil-dwelling Streptomyces might have arisen from evolutionaryspecialization of ActE, perhaps driven by association with theSirex-fungal symbiosis.

ActE contained 12 CAZy families not found in the other modelcellulolytic organisms shown in FIG. 3, including GHs, CBMs, and PLs.Seven other CAZy categories, primarily hemicellulases, were shared onlywith T. reesei. ActE had 23 GH, 10 CBM and 2 PL not found inThermobifida fusca, another cellulolytic Actinomycetales, which had only1 GH and 1 CBM not found in ActE. The genome sequence revealed C.japonicus (strain Ueda 107) is highly enriched in GH43 enzymes requiredfor hemicellulose utilization, but is missing a key reducing endexocellulase (bacterial GH48) required for robust growth on cellulose[e.g., see page 5459 of (DeBoy et al., 2008)]; both of these enzymefamilies are present in highly cellulolytic ActE. Furthermore, ActE alsocontained 6 genes from the CBM33 family, recently shown to catalyzeoxidative cleavage of chitin (Vaaje-Kolstad et al., 2010) and cellulose(Forsberg et al., 2011). Thus, ActE has genomic composition overlappingother cellulolytic organisms, but with notable expansion in the CAZycomposition for both hydrolytic and oxidative enzymes and the presenceof the complete set of enzymes required for efficient cellulosedeconstruction.

Example 4 Genome-Wide Gene Expression Analysis of ActE CAZy Gene

Gene expression profiles were determined for ActE grown on purifiedpolysaccharides and plant biomass by whole genome microarrays (FIGS. 4and 5, FIGS. 9 to 14). Genome-wide gene expression was analyzed as afunctional annotation network composed of ActE genes (circles) connectedto predicted functional groups (triangles; KEGG or CAZy). In FIG. 4, thenetwork was annotated with genome-wide microarray expression data toindicate genes that were differentially expressed when ActE was grown oneither AFEX-SG or glucose, and further annotated to indicate normalizedexpression levels observed during growth on AFEX-SG. While many aspectsof metabolism are modestly changed in response to these different carbonsources, the CAZy and ABC transporter categories were substantiallyenriched in differentially expressed genes (FIG. 4, green circles).Furthermore, pentose sugar metabolism, sulfur metabolism, and some aminoacid biosynthesis pathways (e.g., aromatic amino acids) were also highlyinduced during growth on AFEX-SG relative to other carbon sources (FIGS.9-14). In contrast, ribosomal, secondary metabolite, and DNA repairgenes showed little change in expression across the conditions examined.Within the CAZy functional group, there was a large induction of genesthat contained both a GH domain and a CBM2 domain. Among the 11 genes inthe ActE genome that contain a CBM2 domain, 6 were induced greater than4-fold during growth on AFEX-SG. Furthermore, 9 of the 11 CBM2containing proteins were identified in the secreted proteome (FIG. 3).

Example 5 ActE CAZy Gene Expression is Dependent on ActE GrowthSubstrate

Given the large number of differentially expressed CAZy genes identifiedin the network analysis, Applicants analyzed the expression of thisgroup of genes in cultures grown on different carbon sources (FIG. 5,FIG. 15 and FIG. 16). As with other cellulolytic organisms, there wasstrong correlation between the content of the secreted proteomes and themost highly expressed genes. Of the 167 ActE genes containing CAZydomains, 68 genes (FIG. 5, group 1) showed distinct increases inexpression when grown on different polymeric substrates, 14 genes (FIG.15, group 2) did not show any appreciable level of expression, and 85genes (FIG. 16, group 3) showed moderate changes in expression with thedifferent substrates. A significant fraction of these genes containedtranslocation signals for either the Sec or twin-arginine translocationpathways, and genes encoding structural polypeptides for thesetranslocation pathways were also highly expressed. Besides correlationwith secreted proteins, the transcriptomic studies also gave insightinto co-regulated gene clusters that potentially encode functional unitsfor utilization of different polysaccharides by ActE. In the following,the 130 genes with normalized expression intensities in the top 2% ofall genes are described.

During growth on cellulose, four CAZy genes (SACTE_(—)0236,SACTE_(—)0237, SACTE_(—)3159, and SACTE_(—)0482) showed >15-foldincrease in transcript abundance (FIG. 5), and the correspondingproteins were highly enriched in the secreted proteome. None of thesefour were obviously placed in a gene cluster, and the two most highlyexpressed genes, SACTE_(—)0236 and SACTE_(—)0237, while adjacent on thechromosome, were transcribed in opposite directions. Nevertheless, thesefour most highly expressed genes and three others that showed >5-foldincrease in transcript abundance (SACTE_(—)3717, SACTE_(—)6428,SACTE_(—)2347, Table 3) were associated with a conserved 14 bppalindromic promoter sequence, TGGGAGCGCTCCCA (the CebR bindingelement). CebR proteins are Lacl/GalR-like transcriptional regulatorsshown to provide transcriptional control of gene expression in responseto the presence of cellobiose or other small oligosaccharides in S.griseus, S. reticuli, and Thermobifida fusca (Marushima, Ohnishi, etal., 2009; Water and Schrempf, 1996; Deng and Fong, 2010). Likewise, thegenes (SACTE_(—)2285 to SACTE_(—)2289) encoding a CebR regulator(SACTE_(—)2285), a GH1 protein (β-glucosidase), a two-protein cellobiosetransporter system, and an extracellular solute binding protein wereassociated with a CebR binding element and were also among the mosthighly expressed genes during growth on cellulose. These latter fivegenes have 75% or greater sequence identity with the cellobioseutilization operon identified in S. griseus and S. reticuli (Marushima,Ohnishi, et al., 2009; Schlosser and Schrempf, 1996). There were only 15genes annotated as hypothetical or domain of unknown function (12%)up-regulated during growth on cellulose, a considerably smallerpercentage of these than in the entire genome (27%).

TABLE 3 Analysis of upstream DNA sequence elements in ActE genesupregulated during growth on cellulose. Catalytic Fold Locus domain CBMAnnotated function Sequence^(a) Rank^(b) change^(b) SACTE_0236 GH48 CBM21,4-beta TGGGAGCGCTC 1 21.7 cellobiohydrolase CCA SACTE_0237 GH6 CBM21,4-beta TGGGAGCGCTC 2 17.3 cellobiohydrolase CCA SACTE_3159 CBM33 CBM2Cellulose-binding TGGGAGCGCTC 3 16.2 domain CCA SACTE_0482 GH5 CBM2Endo-1,4-beta- TGGGAGCGCTC 4 15.4 glucosidase CCA SACTE_2288Transport systems TGGGAGCGCTC 5 11.2 inner membrane CCA componentSACTE_3717 GH9 CBM2 1,4-beta TGGGAGCGCTC 6 9.7 cellobiohydrolase CCASACTE_6428 CBM33 Chitin-binding, GGGAGCGCTCC 9 7.9 domain 3 CASACTE_2347 GH5 CBM2 Beta-mannosidase TGGGAGCGCTC 11 5.0 CCA SACTE_2287Transport systems TGGGAGCGCTC 15 4.3 inner membrane CCA componentSACTE_2289 Family 1 TGGGAGCGCTC 19 3.9 extracellular CCA solute-bindingprotein SACTE 0352 GCN5-related N- TGGGAGCGCTC 22 3.6 acetyltransferaseCCA SACTE_2286 GH1 Glycoside GGGAGCGCTCC 27 3.4 hydrolase 1 CASACTE_0483 CBM2  Cellulose-binding GGGAGCGCTCC 503 1.6 family protein CASACTE_0562 GH74 CBM2 Secreted cellulase TGGGAGCGCTC 5759 0.7 (endo) CCASACTE_2285 Lacl family TGGGAGCGCTC 6229 0.6 transcriptional CCAregulator (CebR) ^(a)Predicted binding sequence element found upstreamfrom gene locus. ^(b)Ranking and fold change in expression intensitydetected by microarray for ActE genes when grown on cellulose relativeto glucose.

Several characteristics distinguished expression during growth on eitherxylan or chitin. First, unique sets of genes were induced, as there wasonly 14% and 10% overlap, respectively, when compared to cellulose.Second, ˜33% of the top 2% of genes expressed during growth on eitherxylan or chitin were annotated as hypothetical or domain of unknownfunction, which greatly exceeds the unknown fraction in the cellulosesecretome. During growth on xylan, two clusters of genes wereup-regulated. One extended from SACTE_(—)0357 to SACTE_(—)0370, encodingproteins from the GH11, GH13, GH42, GH43, GH78, GH87, and CE4 families,a Lacl-like transcriptional regulator, a secreted peptidase, and twosets of inner membrane transporters and associated solute bindingproteins. Alternatively, during growth on chitin, three CBM33 proteinswere up-regulated (SACTE_(—)0080, SACTE_(—)2313, SACTE_(—)6493), and twoof these had an immediately adjacent gene encoding a GH18(SACTE_(—)6494) or GH19 (SACTE_(—)0081) that was up-regulated.

When ActE was grown on biomass samples, 14 additional CAZy genes wereuniquely up regulated, and the corresponding proteins were identified inthe proteomic analysis of biomass secretomes (FIGS. 3 and 4). A genecluster extending from SACTE_(—)5858 to SACTE_(—)5864 was uniquely upregulated during growth on biomass. Among these genes, SACTE_(—)5860 andSACTE_(—)5862 are annotated as a twin-arginine translocation pathwayprotein and an ABC transporter, respectively, while the rest areannotated either as hypothetical protein or as domain of unknownfunction.

Eight CAZy genes were >4-fold up-regulated during growth on cellulose,including endoglucanases, reducing and non-reducing end exoglucanases,xylanase and CBM33 proteins (FIG. 5, Table 4). During growth on xylan,eight CAZy genes were elevated >4-fold relative to glucose, includingexoglucanase, xylanase, pectate lyase and other hemicellulases (Table4). Furthermore, chitin-grown cells contained 2 up-regulated genes fromCAZy families including chitinase (SACTE_(—)4571) and a CBM33 protein[SACTE_(—)2313, an ortholog of oxidative chitin oxidase from S.marcescens (Vaaje-Kolstad et al., 2010)]. Thus on a genome-wide basisActE selectively expresses small, distinct sets of CAZy genes duringgrowth on pure polysaccharides, which is distinct from the largernumbers of CAZy genes expressed by T. reesei (Herpoel-Gimbert et al.,2008), C. thermocellum (Raman et al., 2009; Riederer et al., 2011), andT. fusca (Chen and Wilson, 2007).

TABLE 4 Streptomyces sp. ActE genes with >4-fold expression increaseduring growth on pure polysaccharides. Fold increase CAZy AnnotationSigmacell: glc xylan: glc chitin: glc Sigmacell SACTE_6428 CBM33Chitin-binding, domain 3 7.06 1.64 1.81 SACTE_3159 CBM33, 2Cellulose-binding domain, 13.03 1.90 1.29 family II, bacterial typeSACTE_0358 GH11, CBM60, 36 Glycoside hydrolase, family 6.28 4.01 2.1211, active site SACTE_0236 GH48, CBM2, 37 Glycoside hydrolase, 48F 19.004.93 3.91 SACTE_0482 GH5, CBM2 Cellulose-binding family 11.84 3.01 2.00II/chitobiase, carbohydrate- binding domain SACTE_2347 GH5, CE3, CBM2,37 Cellulose-binding family 4.46 1.17 0.99 II/chitobiase, carbohydrate-binding domain SACTE_0237 GH6, CBM2 1,4-beta cellobiohydrolase 15.331.12 0.77 SACTE_3717 GH9, CBM4, 2 Carbohydrate-binding, 8.03 2.61 1.55CenC-like SACTE_2288 Binding-protein-dependent 11.05 4.76 3.26 transportsystems inner membrane component SACTE_0168 Transcription regulatorLuxR, 7.55 1.53 1.37 C-terminal SACTE_0169 Glyceraldehyde 3-phosphate5.01 0.75 1.08 dehydrogenase, active site SACTE_3594 Peptidase S1C, 4.523.36 2.70 HrtA/DegP2/Q/S SACTE_5228 Binding-protein-dependent 4.20 4.353.24 transport systems inner membrane component Xylan SACTE_4029 CE4Glycoside 1.07 4.35 2.22 hydrolase/deacetylase, beta/alpha-barrelSACTE_0358 GH11, CBM60, 36 Glycoside hydrolase, family 6.28 4.01 2.1211, active site SACTE_0382 GH2, CBM42 Galactose-binding domain- 1.794.18 2.46 like SACTE_1230 GH23 Lytic transglycosylase-like, 1.29 5.643.70 catalytic SACTE_0816 GH31 Glycoside hydrolase, family 1.53 4.513.27 31 SACTE_0236 GH48, CBM2, 37 Glycoside hydrolase, 48F 19.00 4.933.91 SACTE_1290 GH53, CBM61 Galactose-binding domain- 1.43 4.73 2.40like SACTE_5978 PL1, CBM35 Galactose-binding domain- 2.00 6.86 2.12 likeSACTE_5325 Binding-protein-dependent 1.78 8.26 3.76 transport systemsinner membrane component SACTE_6023 Galactose-binding domain- 1.92 7.843.34 like SACTE_1834 Alkaline phosphatase D- 1.78 7.73 3.98 relatedSACTE_6100 Sulfate transporter 2.07 7.45 4.75 SACTE_5361 hypotheticalprotein 1.77 7.20 3.94 SACTE_5163 Lambda repressor-like, DNA- 1.47 6.893.29 binding SACTE_6365 Isocitrate 1.88 6.82 4.01 lyase/phosphorylmutaseSACTE_0254 Thiolase-like 2.13 6.76 5.02 SACTE_6478 FAD-dependentpyridine 2.00 6.72 4.46 nucleotide-disulfide oxidoreductase SACTE_3570hypothetical protein 1.61 6.71 3.72 SACTE_0590 Polyketide 1.55 6.67 4.42cyclase/dehydrase SACTE_3152 Twin-arginine translocation 1.41 6.60 2.98pathway, signal sequence SACTE_5285 Bacterial bifunctional 1.71 6.543.33 deaminase-reductase, C- terminal SACTE_1383 Glycerophosphoryldiester 1.08 6.50 3.51 phosphodiesterase SACTE_4333Binding-protein-dependent 1.37 6.46 3.58 transport systems innermembrane component SACTE_3876 hypothetical protein 1.21 6.42 2.73SACTE_6340 Monooxygenase, FAD- 2.82 6.27 3.69 binding SACTE_4237hypothetical protein 1.82 6.27 2.91 SACTE_5136 NAD(P)-binding domain2.20 6.27 2.87 SACTE_6561 hypothetical protein 2.92 6.06 5.65 SACTE_0686Transcription regulator 0.88 6.04 2.72 AsnC-type SACTE_0817 NUDIXhydrolase, conserved 1.96 6.03 3.19 site SACTE_3004 Type II secretionsystem F 1.67 6.01 4.18 domain SACTE_1835 DoxX 1.66 5.97 3.30 SACTE_1933hypothetical protein 0.93 5.96 2.77 SACTE_6290 Glyoxalase/bleomycin 1.865.95 4.10 resistance protein/dioxygenase SACTE_5583 hypothetical protein1.33 5.87 4.56 SACTE_0586 hypothetical protein 1.40 5.81 2.90 SACTE_0046NADH: flavin 2.48 5.75 4.56 oxidoreductase/NADH oxidase, N-terminalSACTE_1096 Mandelate 1.19 5.73 3.32 racemase/muconate lactonizingenzyme, N- terminal SACTE_2897 hypothetical protein 1.18 5.73 3.81SACTE_5359 Rhs repeat-associated core 1.30 5.70 2.41 SACTE_0200hypothetical protein 1.34 5.67 3.64 SACTE_0018 hypothetical protein 1.675.63 3.58 SACTE_5542 hypothetical protein 2.03 5.61 3.52 SACTE_3137hypothetical protein 1.46 5.61 3.91 SACTE_0017 DNA helicase, UvrD/REP2.32 5.58 4.26 type SACTE_0672 hypothetical protein 1.53 5.54 3.20SACTE_1393 Urease, beta subunit 2.08 5.53 3.67 SACTE_0064 Transcriptionregulator PadR 2.17 5.52 3.07 N-terminal-like SACTE_1168 PeptidaseS1/S6, 0.98 5.51 3.36 chymotrypsin/Hap SACTE_6371 hypothetical protein1.37 5.51 3.44 SACTE_4334 Binding-protein-dependent 1.46 5.50 3.35transport systems inner membrane component SACTE_2457 CDP-glycerol 1.075.48 3.79 glycerophosphotransferase SACTE_4734 Binding-protein-dependent1.21 5.44 3.31 transport systems inner membrane component SACTE_3661hypothetical protein 1.76 5.44 3.25 SACTE_0036 hypothetical protein 1.755.43 2.99 SACTE_6005 Citrate synthase-like, core 1.01 5.38 2.90SACTE_6562 hypothetical protein 2.34 5.36 3.37 SACTE_1937 Majorfacilitator superfamily 0.88 5.34 3.02 MFS-1 SACTE_6220 Dodecinflavoprotein 2.13 5.32 5.08 SACTE_0778 FMN-binding split barrel 1.135.28 2.72 SACTE_5672 Acyltransferase 3 1.33 5.28 3.09 SACTE_5989Cysteine-rich domain 1.40 5.24 3.11 SACTE_5296 HTH transcriptional 1.425.22 2.96 regulator, MarR SACTE_2021 hypothetical protein 1.44 5.17 2.54SACTE_1845 Transposase, IS4-like 1.69 5.16 3.30 SACTE_1771 Phage T4-likevirus tail tube 1.55 5.10 1.71 gp19 SACTE_2583 hypothetical protein 1.385.10 3.11 SACTE_5957 Helix-turn-helix, HxIR type 2.38 5.09 3.95SACTE_4642 hypothetical protein 1.31 5.08 3.05 SACTE_3695Aminoglycoside/hydroxyurea 1.41 5.03 3.76 antibiotic resistance kinaseSACTE_0079 ATPase-like, ATP-binding 2.21 5.01 2.98 domain SACTE_0727hypothetical protein 2.54 5.00 3.88 SACTE_0019 hypothetical protein 1.375.00 2.40 SACTE_6422 Streptomyces 2.40 4.99 3.57 cyclase/dehydraseSACTE_4348 Bacterial extracellular solute- 1.60 4.97 3.06 bindingprotein, family 5 SACTE_5318 Forkhead-associated (FHA) 1.50 4.93 2.84domain SACTE_5413 Urease accessory protein 1.94 4.93 2.52 UreFSACTE_5434 Glutathione S-transferase, 2.41 4.93 2.96 C-terminal-likeSACTE_6061 Glyoxalase/bleomycin 1.61 4.92 2.18 resistanceprotein/dioxygenase SACTE_0025 hypothetical protein 1.58 4.92 4.22SACTE_5552 Transposase, IS4-like 1.94 4.92 3.26 SACTE_4156 HTHtranscriptional 1.57 4.86 2.81 regulator, LysR SACTE_5600 hypotheticalprotein 1.78 4.83 2.01 SACTE_5331 Conserved hypothetical 1.56 4.82 2.96protein CHP03086 SACTE_0784 hypothetical protein 1.43 4.80 2.65SACTE_0045 NAD(P)-binding domain 1.74 4.78 3.35 SACTE_5426 Twin-argininetranslocation 0.80 4.77 2.68 pathway, signal sequence SACTE_2654 4Fe—4Sferredoxin, iron- 1.30 4.77 2.68 sulfur binding domain SACTE_2288Binding-protein-dependent 11.05 4.76 3.26 transport systems innermembrane component SACTE_2324 Membrane insertion protein, 0.91 4.75 2.58OxaA/YidC, core SACTE_0142 Amidohydrolase 2 1.28 4.71 2.65 SACTE_0787hypothetical protein 1.66 4.70 2.93 SACTE_5790 hypothetical protein 1.284.69 2.83 SACTE_6291 hypothetical protein 1.25 4.68 3.13 SACTE_6499hypothetical protein 1.66 4.67 3.29 SACTE_6548 Lytictransglycosylase-like, 1.97 4.66 3.20 catalytic SACTE_3087 Majorfacilitator superfamily 1.30 4.66 3.26 MFS-1 SACTE_5512 hypotheticalprotein 1.79 4.64 3.48 SACTE_0491 hypothetical protein 2.44 4.63 2.71SACTE_0312 Thiamine pyrophosphate 2.32 4.60 3.49 enzyme, C-terminal TPP-binding SACTE_6130 hypothetical protein 1.47 4.55 2.64 SACTE_3787Helix-turn-helix type 3 1.38 4.53 2.73 SACTE_0040 hypothetical protein1.64 4.52 4.80 SACTE_2461 Macrocin-O- 1.07 4.51 3.00 methyltransferaseSACTE_5041 hypothetical protein 1.50 4.49 3.25 SACTE_5540 Transposase,1.79 4.49 2.99 IS204/IS1001/IS1096/IS1165 SACTE_0776 Protein of unknownfunction 1.34 4.48 2.52 DUF6, transmembrane SACTE_0785 Bacterial TniB1.67 4.43 2.93 SACTE_0360 Binding-protein-dependent 1.70 4.43 2.39transport systems inner membrane component SACTE_3569 Protein of unknownfunction 1.00 4.42 2.78 DUF1023 SACTE_2986 hypothetical protein 1.624.42 2.96 SACTE_4732 Twin-arginine translocation 2.08 4.41 2.72 pathway,signal sequence SACTE_5228 Binding-protein-dependent 4.20 4.35 3.24transport systems inner membrane component SACTE_0406Binding-protein-dependent 1.34 4.35 2.52 transport systems innermembrane component SACTE_6516 Binding-protein-dependent 2.24 4.34 3.41transport systems inner membrane component SACTE_1781 hypotheticalprotein 1.16 4.34 2.56 SACTE_5936 Radical SAM 1.43 4.33 2.23 SACTE_0819Protein of unknown function 1.50 4.33 2.83 DUF962 SACTE_4539 NERD 1.424.32 3.98 SACTE_0532 Binding-protein-dependent 3.47 4.31 2.42 transportsystems inner membrane component SACTE_3300 hypothetical protein 1.684.31 2.59 SACTE_6277 hypothetical protein 2.24 4.31 3.11 SACTE_0941Twin-arginine translocation 1.32 4.30 2.63 pathway, signal sequenceSACTE_1115 GntR, C-terminal 1.57 4.29 2.63 SACTE_6105 Fatty acidhydroxylase 1.63 4.29 2.78 SACTE_4407 Spherulation-specific family 41.19 4.29 4.15 SACTE_5387 hypothetical protein 1.24 4.27 3.08 SACTE_5053NmrA-like 1.23 4.27 3.05 SACTE_5562 Amino acid ABC transporter, 1.374.26 3.75 permease protein, 3-TM domain, His/Glu/Gln/Arg/opine familySACTE_5522 Galactose-binding domain- 1.82 4.26 2.62 like SACTE_5484Transcription regulator, 1.45 4.21 3.24 TetR-like, DNA-binding,bacterial/archaeal SACTE_6526 Restriction endonuclease, 2.31 4.20 2.40type IV-like, Mrr SACTE_4164 hypothetical protein 1.06 4.19 2.48SACTE_4979 Transcription regulator, 1.20 4.19 2.34 TetR-like,DNA-binding, bacterial/archaeal SACTE_0952 hypothetical protein 1.334.18 2.02 SACTE_1785 hypothetical protein 1.25 4.17 1.94 SACTE_3454hypothetical protein 1.46 4.16 2.32 SACTE_1271 Class IIaldolase/adducin, N- 1.77 4.16 2.65 terminal SACTE_1760 hypotheticalprotein 1.38 4.13 2.07 SACTE_0035 hypothetical protein 1.93 4.13 3.13SACTE_0247 Protein of unknown function 1.30 4.10 2.77 DUF2241 SACTE_3796F420-dependent enzyme, 1.43 4.10 3.33 PPOX class, family Rv2061,putative SACTE_4641 hypothetical protein 1.43 4.09 2.60 SACTE_4816Peptidase S26, conserved 1.17 4.09 2.77 region SACTE_2331 Majorfacilitator superfamily 1.15 4.08 2.20 MFS-1 SACTE_1666 hypotheticalprotein 1.44 4.07 2.46 SACTE_5867 Mammalian cell entry, 1.79 4.07 2.92mce1C SACTE_2705 AMP-binding, conserved site 1.38 4.07 2.75 SACTE_6014Binding-protein-dependent 0.89 4.07 2.51 transport systems innermembrane component SACTE_2018 Putative DNA binding 1.05 4.06 2.63 domainSACTE_5690 Gluconate transporter 1.00 4.05 2.29 SACTE_3243 hypotheticalprotein 0.91 4.05 2.23 SACTE_0786 Polynucleotidyl transferase, 1.81 4.032.98 ribonuclease H fold SACTE_6450 Rhamnose isomerase 2.72 4.02 2.90related SACTE_0097 Beta-lactamase-related 1.70 4.02 2.52 SACTE_6341FMN-binding split barrel, 1.82 4.01 2.45 related SACTE_1483 hypotheticalprotein 0.82 4.01 2.75 SACTE_0754 Uncharacterised protein 1.21 4.00 2.51family UPF0060 SACTE_5308 Winged helix-turn-helix 1.33 4.00 1.56transcription repressor DNA- binding SACTE_5862 ABC transporter,conserved 1.87 4.00 3.05 site Chitin SACTE_2313 CBM33 Chitin-binding,domain 3 1.08 1.24 4.77 SACTE_4571 GH18, CBM57, 2 EF-Hand 1,calcium-binding 0.88 1.37 4.08 site SACTE_5381 hypothetical protein 1.313.09 10.06 SACTE_5386 hypothetical protein 0.96 1.59 8.49 SACTE_1949Peptidase M4, thermolysin 1.30 2.16 7.57 SACTE_6519Binding-protein-dependent 2.00 3.04 7.36 transport systems innermembrane component SACTE_0243 Protein kinase-like domain 1.68 2.55 6.89SACTE_6520 ABC transporter, conserved 1.03 1.18 6.25 site SACTE_5384hypothetical protein 1.16 2.39 5.99 SACTE_6463 hypothetical protein 1.282.52 5.85 SACTE_6561 hypothetical protein 2.92 6.06 5.65 SACTE_5383hypothetical protein 1.06 1.69 5.28 SACTE_6518 hypothetical protein 1.661.91 5.21 SACTE_4797 hypothetical protein 2.22 0.34 5.19 SACTE_6170Domain of unknown function 1.47 3.49 5.12 DUF1996 SACTE_6220 Dodecinflavoprotein 2.13 5.32 5.08 SACTE_0254 Thiolase-like 2.13 6.76 5.02SACTE_2678 Protein of unknown function 1.40 1.13 5.02 DUF397 SACTE_5968hypothetical protein 1.58 1.31 4.90 SACTE_4757 Acetyl-coenzyme A 1.640.59 4.86 carboxyltransferase, C- terminal SACTE_0040 hypotheticalprotein 1.64 4.52 4.80 SACTE_6100 Sulfate transporter 2.07 7.45 4.75SACTE_1833 Twin-arginine translocation 1.64 1.56 4.64 pathway, signalsequence SACTE_5583 hypothetical protein 1.33 5.87 4.56 SACTE_0046 NADH:flavin 2.48 5.75 4.56 oxidoreductase/NADH oxidase, N-terminal SACTE_5398hypothetical protein 1.45 1.73 4.55 SACTE_6144 Twin-argininetranslocation 1.21 1.13 4.52 pathway, signal sequence SACTE_6478FAD-dependent pyridine 2.00 6.72 4.46 nucleotide-disulfideoxidoreductase SACTE_0590 Polyketide 1.55 6.67 4.42 cyclase/dehydraseSACTE_2112 Homeodomain-like 1.44 1.33 4.40 SACTE_0017 DNA helicase,UvrD/REP 2.32 5.58 4.26 type SACTE_5841 Protein of unknown function,1.90 3.09 4.24 ATP binding SACTE_0025 hypothetical protein 1.58 4.924.22 SACTE_3004 Type II secretion system F 1.67 6.01 4.18 domainSACTE_4407 Spherulation-specific family 4 1.19 4.29 4.15 SACTE_0307Protein of unknown function 1.13 1.79 4.15 DUF320, Streptomyces speciesSACTE_6290 Glyoxalase/bleomycin 1.86 5.95 4.10 resistanceprotein/dioxygenase SACTE_5286 hypothetical protein 1.33 3.34 4.07SACTE_5953 Protein of unknown function, 1.35 2.11 4.05 ATP bindingSACTE_6365 Isocitrate 1.88 6.82 4.01 lyase/phosphorylmutase

Example 6 Composition of ActE Secretome is Dependent on ActE GrowthSubstrate

To identify secreted proteins, supernatants from ActE cultures grown onglucose, cellobiose, cellulose, xylan, chitin, switchgrass, AFEX-SG, andIL-SG were analyzed by LC-MS/MS (FIG. 3 and FIG. 18). The proteins weresorted into a descending rank according to spectral counts, and setswhose spectral counts summed to 95% of the total protein in eachsecretome are shown. FIG. 3A summarizes the percentages of CAZy familiesin the detected proteins. The glucose secretome had a proteinconcentration of ˜0.03 g/L of culture medium, and among the 136 proteinsidentified only 3% had a CAZy annotation. Indeed, the majority (>90%)likely originated from cell lysis. In contrast, the polysaccharidesecretomes had a protein concentration of ˜0.3 g/L of culture medium, a˜10-fold increase from the glucose secretome. Pectate lyase(SACTE_(—)1310), chondroitin/alginate lyase (SACTE_(—)4638), anextracellular solute binding protein (SACTE_(—)4343), bacterioferritin(SACTE_(—)1546), and catalase (SACTE_(—)4439) were observed in allpolysaccharide secretomes. The first two proteins, SACTE_(—)1310 andSACTE_(—)4638, have signal peptides and are thus secreted as part of theresponse needed for growth on polysaccharides.

FIG. 3 and FIG. 18 further demonstrate that 22 proteins accounted for95% of the total spectral counts during growth on cellulose; two-thirdswere from CAZy families. The five most abundant proteins, in order andrepresenting ˜85% of the total spectral counts, were reducing andnon-reducing exoglucanases (SACTE_(—)0236 and SACTE_(—)0237), a CBM33polysaccharide monooxygenase (SACTE_(—)3159), an endoglucanase(SACTE_(—)0482), and a β-mannosidase (SACTE_(—)2347). The first fourproteins encode a non-redundant set of enzymes that likely provide theessential activities required for utilization of crystalline cellulose(Deboy, et al., 2008). Among the 22 most abundant proteins, there wererepresentatives from 9 different GH families, two CE families, two PLfamilies, and two additional CMB33 proteins. Collectively, thesesecreted proteins represent ˜20% of the CAZy composition in the ActEgenome.

There were substantial differences in the composition of the xylan andchitin secretomes as compared to the cellulose secretome (FIG. 3 andFIG. 18). In the xylan secretome, 92 proteins comprise 95% of thedetected spectral counts. Twenty GHs from 18 different CAZy familieswere included, along with 1 CE4 and 2 PL family proteins. Thus, growthon xylan elicits secretion of representatives from half of the totalCAZy families found in the ActE genome. The broad distribution ofhemicellulytic enzymes in the xylan secretome contrasts with theconsiderably less diverse composition of the chitin secretome, whichconsists of 7 representatives from GH18 (e.g., chitinase, endobeta-N-acetylglucosaminidase), 2 from GH19 (e.g., chitinase, lysozyme),and 1 chitinolytic CBM33 (FIG. 18). While chitinolytic CAZy familiesaccount for two-thirds of the proteins secreted during growth on chitin,they represent only ˜6% of the diversity of CAZy families found in thegenome. These results document the substantially differentsubstrate-specific responses of ActE during growth on differentpolysaccharides.

The secretomes isolated from cells grown on switchgrass, AFEX-SG, andIL-SG contained the highly abundant secreted proteins identified in thepurified cellulose and xylan experiments and some additional proteins.These additional proteins likely reflect cellular response to the morecomplex composition of polysaccharides present in the biomass samples.The increased diversity of proteins present in the biomass secretomealso increased the efficiency of reaction with plant biomass (FIG. 2C).In total, the biomass secretomes contained 31 different CAZy familiesthat contributed to the total spectral counts (˜70% of the CAZy familiespresent in the ActE genome), thus representing coordinated and extensiveuse of CAZyme families present in the ActE genome for biomassutilization.

The gene loci of the 117 proteins observed only in the glucose secretomeare: SACTE_(—)0494; SACTE_(—)0514; SACTE_(—)0541; SACTE_(—)0548;SACTE_(—)0604; SACTE_(—)0669; SACTE_(—)0687; SACTE_(—)0800;SACTE_(—)0810; SACTE_(—)0899; SACTE_(—)1006; SACTE_(—)1045;SACTE_(—)1068; SACTE_(—)1069; SACTE_(—)1111; SACTE_(—)1201;SACTE_(—)1240; SACTE_(—)1285; SACTE_(—)1328; SACTE_(—)1344;SACTE_(—)1368; SACTE_(—)1419; SACTE_(—)1426; SACTE_(—)1506;SACTE_(—)1522; SACTE_(—)1586; SACTE_(—)1650; SACTE_(—)1861;SACTE_(—)1888; SACTE_(—)1934; SACTE_(—)2036; SACTE_(—)2049;SACTE_(—)2068; SACTE_(—)2238; SACTE_(—)2403; SACTE_(—)2431;SACTE_(—)2468; SACTE_(—)2558; SACTE_(—)2645; SACTE_(—)2729;SACTE_(—)2755; SACTE_(—)2756; SACTE_(—)2801; SACTE_(—)2819;SACTE_(—)3012; SACTE_(—)3037; SACTE_(—)3067; SACTE_(—)3086;SACTE_(—)3088; SACTE_(—)3097; SACTE_(—)3219; SACTE_(—)3327;SACTE_(—)3361; SACTE_(—)3371; SACTE_(—)3385; SACTE_(—)3389;SACTE_(—)3392; SACTE_(—)3414; SACTE_(—)3438; SACTE_(—)3511;SACTE_(—)3604; SACTE_(—)3716; SACTE_(—)3896; SACTE_(—)3948;SACTE_(—)3955; SACTE_(—)3956; SACTE_(—)3960; SACTE_(—)3961;SACTE_(—)3989; SACTE_(—)3995; SACTE_(—)4030; SACTE_(—)4031;SACTE_(—)4038; SACTE_(—)4039; SACTE_(—)4073; SACTE_(—)4081;SACTE_(—)4083; SACTE_(—)4145; SACTE_(—)4191; SACTE_(—)4194;SACTE_(—)4205; SACTE_(—)4224; SACTE_(—)4281; SACTE_(—)4283;SACTE_(—)4376; SACTE_(—)4397; SACTE_(—)4399; SACTE_(—)4415;SACTE_(—)4462; SACTE_(—)4501; SACTE_(—)4550; SACTE_(—)4565;SACTE_(—)4566; SACTE_(—)4567; SACTE_(—)4568; SACTE_(—)4591;SACTE_(—)4610; SACTE_(—)4616; SACTE_(—)4618; SACTE_(—)4652;SACTE_(—)4718; SACTE_(—)4768; SACTE_(—)4791; SACTE_(—)4795;SACTE_(—)4830; SACTE_(—)4860; SACTE_(—)4873; SACTE_(—)4926;SACTE_(—)4959; SACTE_(—)5028; SACTE_(—)5081; SACTE_(—)5192;SACTE_(—)5267; SACTE_(—)5482; SACTE_(—)5519; SACTE_(—)5983; andSACTE_(—)6342.

The gene loci of the 9 proteins observed only in the Sigmacell secretomeare: SACTE_(—)0236; SACTE_(—)0482; SACTE_(—)0562; SACTE_(—)2313;SACTE_(—)2347; SACTE_(—)3590; SACTE_(—)3717; SACTE_(—)4571; andSACTE_(—)6428.

The gene loci of the 46 proteins observed only in the xylan secretomeare: SACTE_(—)0081; SACTE_(—)0169; SACTE_(—)0365; SACTE_(—)0379;SACTE_(—)0383; SACTE_(—)0464; SACTE_(—)0528; SACTE_(—)0549;SACTE_(—)0634; SACTE_(—)0880; SACTE_(—)1003; SACTE_(—)1130;SACTE_(—)1239; SACTE_(—)1324; SACTE_(—)1325; SACTE_(—)1356;SACTE_(—)1364; SACTE_(—)1367; SACTE_(—)1603; SACTE_(—)1680;SACTE_(—)1858; SACTE_(—)1949; SACTE_(—)2768; SACTE_(—)3064;SACTE_(—)4231; SACTE_(—)4246; SACTE_(—)4363; SACTE_(—)4459;SACTE_(—)4483; SACTE_(—)4515; SACTE_(—)4607; SACTE_(—)4612;SACTE_(—)4624; SACTE_(—)4730; SACTE_(—)4755; SACTE_(—)4858;SACTE_(—)5166; SACTE_(—)5230; SACTE_(—)5231; SACTE_(—)5418;SACTE_(—)5457; SACTE_(—)5630; SACTE_(—)5647; SACTE_(—)5682;SACTE_(—)5751; and SACTE_(—)6439.

In the xylan secretome, five proteins accounted for half of the totalsecreted protein. These were xylanases (GH10 and GH11, respectively;SACTE_(—)0265, 9.7% and SACTE_(—)0358, 8.1%), extracellular xyloseisomerase (SACTE_(—)5230, 12.7%), acetyl xylan esterase (CE4;SACTE_(—)0357, 11.7%), and pectate lyase (PL1, SACTE_(—)5978, 6.6%).Among the remaining 98 proteins, there were numerous GH families. Giventhe complexity of hemicellulose, which is enriched in xylan but alsocontains many other sugars and many different bonding linkages betweenthese sugars, it is noted that these additional proteins represent manyGH families associated with unique hemicellulolytic activities.

Although not analyzed in FIG. 34, the chitin secretome contained tenproteins from the chitinase GH18 (49% of total protein) and GH19 (21%)families. In addition, the CBM33 protein SACTE_(—)2313, having 50%primary sequence identity with the CBP21 chitin oxygenase from S.marcescens, was also detected (3.9%). Insect molt and fungal hyphaeprovide abundant chitin, likely accounting for the utility of theseenzymes in the natural environment. There were 50 other proteins (63total) that comprised 95% of the chitin secretome. Relative to theglucose, Sigmacell, and xylan secretomes, the following 15 proteins wereobserved only in the chitin secretome: SACTE_(—)0746, SACTE_(—)0844,SACTE_(—)0860, SACTE_(—)1702, SACTE_(—)2033, SACTE_(—)2059,SACTE_(—)2062, SACTE_(—)2384, SACTE_(—)3685, SACTE_(—)4468,SACTE_(—)4472, SACTE_(—)4727, SACTE_(—)5330, SACTE_(—)5764, andSACTE_(—)6494.

The gene loci of the 19 proteins observed only in the switchgrasssecretome are: SACTE_(—)0642; SACTE_(—)1130; SACTE_(—)1250;SACTE_(—)1858; SACTE_(—)2033; SACTE_(—)3012; SACTE_(—)3777;SACTE_(—)4198; SACTE_(—)4571; SACTE_(—)4624; SACTE_(—)4669;SACTE_(—)4676; SACTE_(—)4718; SACTE_(—)4738; SACTE_(—)5220;SACTE_(—)5418; SACTE_(—)5685; SACTE_(—)5751; and SACTE_(—)5880.

The gene loci of the 8 proteins observed only in the IL-SG secretomeare: SACTE_(—)0132; SACTE_(—)0880; SACTE_(—)2556; SACTE_(—)4246;SACTE_(—)4515; SACTE_(—)4702; SACTE_(—)5231; and SACTE_(—)5330.

There were no proteins observed only in the AFEX-SG secretome whencompared to either the switchgrass or IL-SG secretomes.

Example 7 Minimized Size of ActE Enzymes Increases Specific Activity

When ActE is grown on Sigmacell, AFEX-SG, IL-SG, AFEX-CS, unbleachedlodgepole pine kraft pulp (UBLPKP) or bleached spruce wood kraft pulp(BSKP), the characteristic secretome consists of the proteins thatpermit deconstruction of these substrates into sugars that can be usedfor growth (FIG. 23). Interestingly, ActE is not capable of growing onlodgepole pine pretreated by SPORL, indicating this pretreatmentproduces toxins that inhibit the growth of highly cellulolytic microbes.When ActE is grown on cellobiose, which it does readily and rapidly, itproduces a secretome that is distinct from those obtained from ActEgrown on cellulose, xylan or biomass substrates, demonstrating that ActEhas highly specific responses to different polymeric substances that arepresent in biomass. This behavior is distinct from that observed for T.fusca, another cellulolytic Actinomycete, and from C. thermocellum,where each organism produced similar sets of secreted proteins duringgrowth on either cellulose or cellobiose (Chen and Wilson, 2007;Riederer et al., 2011). This result indicates ActE contains a uniqueregulatory mechanism for controlling cellulose deconstruction genes thatcan provide exquisite control of their production under desiredcircumstances.

For a single enzyme from a secretome, (Segel, Enzyme kinetics: behaviorand analysis of rapid equilibrium and steady state enzyme systems.Wiley, New York, 1993) the specific activity (μmol/min/mg) is defined asmol of product formed per unit time (i.e., μmol/min) per unit mass ofenzyme (i.e., mg). Specific activity is the parameter that must be usedin making comparisons of catalytic properties between enzymes withdifferent molecular masses. If two enzyme isoforms yield the sameμmol/min, the isoform with the smaller molecular weight will, bydefinition, have the higher specific activity. In this application, itis relevant to consider the implications of a 10% or more reduction inthe mass of an enzyme required to treat gigatonnes of biomass.

In the cellulose secretome, five proteins contributed ˜85% of the totalspectral counts. These were reducing and non-reducing end exoglucanases,endoglucanases, and CBM33 (SACTE_(—)0237, SACTE_(—)0236, SACTE_(—)2347,SACTE_(—)0482 and SACTE_(—)3159); xylanase, another endoglucanase, andanother CBM33 were also abundant (SACTE_(—)0265, SACTE_(—)3717 andSACTE_(—)6428). According to the definition provided above, sizeminimization is a way to achieve the desired increases in specificactivity. Interestingly, the set of ActE enzymes described above are onaverage 10% smaller in mass than their closest orthologs from T. fusca(Chen and Wilson, 2007), suggesting size minimization may have occurredin ActE (Table 5). These enzymes also provide all of the requisitecatalytic reactions needed for the deconstruction of crystallinecellulose.

TABLE 5 ActE cellulose secretome proteins and corresponding best matchin T. fusca. The single protein SACTE_0237 is the best match to bothCel6A and Cel6B suggesting one protein from ActE might replace twoproteins from another organism. ActE T fusca Protein Gene locus CAZyresidues MW identity coverage Gene locus name residues MW SACTE_0237 GH6586 49 80 Tfu_1074 Cel6A 441 45844 SACTE_0237 GH6 586 61062 62 93Tfu_0620 Cel6B 596 63548 SACTE_0236 GH48 954 100726 57 95 Tfu_1959Cel48A 984 107127 SACTE_2347 GH8 562 57753 45 23 Tfu_2176 Cel9A 88095203 SACTE_3159 CBM33 362 37787 42 71 Tfu_1665 E8 438 46808 SACTE_0482GH5 456 47654 51 97 Tfu_0901 Cel5A 466 49807 SACTE_0265 GH10 458 4768344 95 Tfu_2923 Xyl10A 491 53185 SACTE_3717 GH9 909 96338 61 82 Tfu_1627Cel9B 998 107045 SACTE_6428 CBM33 222 24668 62 99 Tfu_1268 E7 222 25372Average identity, coverage 53 82 Sum ActE 4509 473671 with Cel6A 4920530391 4509 473671 with Cel6B 5075 548095 Percentage with Cel6A 92% 89%with Cel6A, B 5516 593939 Percentage with Cel6B 89% 86% Percentage withCel6A, B 82% 80%

Example 8 ActE Secretome Specific Activity is Comparable to that ofSpezyme CP™

The enzymatic activities of ActE secretomes were compared with acommercial secretome, SPEZYME CP. The enzyme cocktail of SPEZYME CP wasprepared from T. reesei Rut-C30, thus providing a useful, routinelyavailable reference point for the capabilities of other cellulolyticorganisms. HPLC analysis showed that the ActE cellulose secretomereleased cellobiose as the primary product during reaction withcellulose (FIG. 2A, 95% of products), which is distinct from the higherproportion of glucose produced by the T. reesei secretome. Similarly,the primary products from xylan and mannan were xylobiose andmannobiose, respectively. Upon accounting for total glucose equivalentsreleased, the ActE secretome obtained from growth on pure cellulose hadspecific activity that was about half of that provided by SPEZYME CP(FIG. 2A, inset). Interestingly, the ActE secretome obtained from growthon pure cellulose had higher specific activity for deconstruction ofpure mannan than SPEZYME CP (FIG. 2B). Additionally, the ActE secretomeobtained from growth on pure xylan had higher specific activity forreaction with pure xylan than SPEZYME CP. Cellulose, xylan, and mannanare all abundant in pinewood, thus accounting for the necessity of eachof the major catalytic activities detected.

Anion exchange chromatography was performed to fractionate the ActEsecretome obtained from cells grown on cellulose as the sole carbonsource. We identified fractions that hydrolyzed pure polysaccharides bybiochemical assays (FIG. 7), and confirmed the identity of the proteinor proteins contained in these fractions by mass spectrometry (FIG. 17).Where multiple polypeptides were present, the identity of each wasconfirmed by mass spectrometry to correspond to the indicated genelocus. In several cases, these most likely arise from proteolysis of asingle protein found in the secretome. Fractions containing the maximumcellulase activity were highly enriched in SACTE_(—)0236 andSACTE_(—)0237, reducing and non-reducing end cellobiohydrolases from theGH6 and GH48 families, respectively. SACTE_(—)0265 and SACTE_(—)2347were identified as the major proteins present in fractions associatedwith xylan and mannan hydrolysis, respectively. A CBM33 polysaccharidemonooxygenase (SACTE_(—)3159) was also identified in the ion exchangeprofile. Moreover, beta-1,3 glucanase activity was identified infractions that were enriched in SACTE_(—)4755.

When ActE was grown on either ammonia fiber expansion-treatedswitchgrass (AFEX-SG) (Li, C. et al., 2011) or ionic liquid-treatedswitchgrass (IL-SG), the secretomes had ˜2-fold increase in specificactivity relative to the cellulose secretome and were equivalent toSPEZYME CP for reaction with both the AFEX- and IL-treated biomass (FIG.2C) (Li, C. et al., 2011). The ActE secretomes retained greater than 60%of maximal activity for the hydrolysis of AFEX- and IL-SG from 30 to 55°C. and 35 to 47° C., respectively, which is comparable to recent reportson the temperature profile of secretomes from thermophilicbiomass-degrading fungi (Tolonen et al., 2011) (FIG. 8A). The secretomesshowed a pH optimum of ˜7 for reaction with AFEX-SG and a pH optimum of˜8 for reaction with IL-SG. Moreover, these secretomes retained greaterthan 60% of maximal activity over the ranges of pH 4.5 to 8.0, and pH7.0 to 8.0, respectively (FIG. 8B). These optimal pH values areconsiderably higher than observed for SPEZYME CP.

Example 9 ActE Produces Cellobiose as the Primary Extracellular Productof Cellulose Utilization

The isolated ActE secretomes contained substantial ability to releasereducing sugars from pure polysaccharides. Cellobiose accounted for ˜95%of soluble sugar released from pure cellulose and glucose representedthe remainder; cellotriose and cellobionic acid were not detected.Neither cellobiosidase nor β-glucosidase was detected in the ActEsecretome. Thus ActE produces cellobiose as the primary extracellularproduct of cellulose utilization and also grows vigorously on this.Dominance of cellobiose may help to channel cellulolytic activity toonly a subset of the Sirex community. Since genes encoding cellobioseoxidase and cellobiose dehydrogenase (Eastwood et al., 2011; Langston etal., 2011) were not present in ActE, biological reduction systems forthe CBM33 proteins may be provided by other members of the Sirexcommunity, in analogy to that described for the heterologous complex ofT. aurantiacus GH61 and Humicola insolens cellobiose dehydrogenase(Langston et al., 2011).

Example 10 Enzymatic Activity of the ActE Secretome can be Improved byAdding One or More Enzymes from Other Organisms or Sources

In the ActE secretome, enzymes SACTE_(—)0236, SACTE_(—)0237, andSACTE_(—)3717 (GH48, GH6, and GH9, respectively) showed decreases incontent of the native forms over a 24 h period, and SACTE_(—)0236 andSACTE_(—)0237 were converted into ˜50 kDa fragments (FIG. 24).SACTE_(—)0359 (CBM33) also showed a time-dependent decrease. Thereactions could be slowed but not eliminated by addition ofphenylmethylsulfonyl fluoride (1 mM), a possible inhibitor of serineproteases (Turini et al., 1969), but not by EDTA (10 mM), a possibleinhibitor of metalloproteases (Trop and Birk, 1970).

SACTE_(—)5668, a serine protease, was detected in all purepolysaccharide secretomes (FIG. 3), while another metallopeptidase,SACTE_(—)3389 annotated as peptidase M24B, X-Prodipeptidase/aminopeptidase P, was detected in all secretomes at lowlevel (0.026%). The protease SACTE_(—)5530 (peptidase S1/S6,chymotrypsin/Hap, 0.1%) was also present in all polysaccharide andbiomass samples. The proteases SACTE_(—)5668 (annotated secretedpeptidase, 0.3%) and SACTE_(—)4231 (serine/cysteine peptidase,trypsin-like, 0.039%) were also detected in all pure polysaccharidesecretomes, and the protease SACTE_(—)6303 (serine/cysteine peptidase,trypsin-like, 0.039%) was also present in all biomass samples.Elimination of one or more of these proteases may impart stabilizationof the enzymatic activity in the secreted proteome.

Addition of CelLcc_CBM3a, an engineered exoglucanase (FIG. 22) thatproduces cellobiose with low specific activity alone (FIG. 21), gave asynergistic increase in the activity of the ActE cellulose secretome.This result demonstrates the potential for heterologous supplementationof the ActE secretome to improve its performance by replacing an enzymeactivity that is lost to proteolysis.

Example 11 ActE Cellulolytic Activity Requires a Minimal Set of Enzymes

When the ActE secretome obtained from growth on cellulose wasfractionated by ion exchange chromatography (FIG. 7), several fractionswere obtained that could be tested in unary, binary, ternary andquaternary combinations for reconstitution of cellulose hydrolysis andother enzymatic activities (FIG. 25). SDS PAGE and LC-MS/MS analysisshowed that these fractions contained the following polypeptides in theapproximate weight percentages: fraction 1, SACTE_(—)3159 (CBM33/CBM2oxidative endocellulase, 95%) and SACTE_(—)4738 (GH16 β-1,3endoglucanase, 5%); fraction 2, SACTE_(—)0237 (GH6 non-reducing endexocellulase, 60%), SACTE_(—)0482 (GH5 endocellulase, 25%),SACTE_(—)4755 (GH64 β-1,3 glucanase, 10%) and SACTE_(—)3159 (oxidativeendocellulase, <5%); and fraction 3, SACTE_(—)0236 (GH48 reducing endexocellulase, 75%), SACTE_(—)3717 (GH9 endocellulase, 20%) andSACTE_(—)5457 (GH46 chitinase, 5%). These results demonstrate thatSACTE_(—)3159 (oxidative endocellulase) provides a complementaryactivity to SACTE_(—)0482 and SACTE_(—)3717 (hydrolytic endocellulolyticactivity). Evidently, the oxidative reaction provides breaks in thecellulose strands that can be readily used by non-reducing and reducingend exocellulases also present in the secretome to processivelydeconstruct the polymeric material.

According to the current understanding of reactions required forhydrolysis of crystalline cellulose, SACTE_(—)3159 (CBM33/CBM2 oxidativeendocellulase), SACT_(—)0482 (GH5), and SACTE_(—)3717 provideendocellulolytic activities, while SACTE_(—)0237 (GH6) providesnon-reducing end exocellulase reaction and SACTE_(—)0236 (GH48) providesreducing end exocellulase activity.

FIG. 16 shows that the secretome contains beta-1,3 endoglucanaseactivity. The majority of this activity corresponds to the fractionscontaining SACTE_(—)4738 and SACTE_(—)4755. These enzymes hydrolyzecallose, a cellulose-like material that is typically produced by plantsin respond to wounding by invasive insects and other trauma.

The proteins described here constitute a naturally evolved and matchedset specialized for the hydrolysis of cellulosic substrates.

Example 12 ActE Mannanase Specific Activity Increases as MannanaseMolecular Weight Decreases

FIG. 26 shows that the mannanase activity present in the ActE secretomeis associated with fractions containing various naturally truncatedvariants of SACTE_(—)2347 (GH5) with molecular weights of ˜57, ˜45, and˜37 kDa. Fractions F9 through F1 from ion exchange chromatographicseparation of the ActE secretome were examined for mannan-deconstructionactivity by Zymogram assay. The basis of the Zymogram assay is asfollows: Congo Red stain interacts with the polysaccharide fraction(mannan) incorporated into the gel and imparts a red color. When anenzyme's activity hydrolyzes the mannan, the interaction of Congo Redwith the polysaccharide is broken and the gel takes on a dark greyappearance. Of note, the strongest mannanase activity was observed infraction F1, which primarily contains the 37 kDa truncated variant.Corresponding to the definition of specific activity given above, the 37kDa variant has an ˜35% increase in specific activity relative to the 57kDa variant. This provides a naturally produced example of how sizereduction may contribute to increased specific activity of enzymes.

Example 13 Recombination of ActE Secretome Fractions ProvidesSynergistic Cellulolytic Activity

FIG. 25 shows synergy of reaction obtained by recombining fractionsobtained from ion exchange fractionation. In FIG. 25A, reactions wereobtained from combinations of the fractions indicated by stars in FIG.27 and FIG. 28. Combination of fractions E5 (oxidative endocellulase)and E11 (hydrolytic endo- and exocellulases) gave a ˜30% increase inproduct yield over that expected from the arithmetic sum of reactions ofE5 and E11 alone, i.e., synergy in reaction. Combination of fractionsE5, E11 and F10 (hydrolytic endo- and exocellulases) gave ˜60% increasein reactivity. In FIG. 25B, reactions were obtained from recombiningfractions shown in FIG. 16. Titration of fraction B1 (full-lengthoxidative endocellulase) into D15 (hydrolytic endo- and exocellulases)shows an optimal reactivity at ˜1:1 ratio of proteins from the twofractions, while an excess of B1 relative to D15 causes decrease inreaction because of depletion of required exocellulase activities.Titration of fraction C4 (truncated oxidative endocellulase andbeta-1,3-endocellulase) with D15 gave maximal stimulation (62% increase)at an 80:20 proportion. These results indicate both forms of oxidativeendocellulase SACTE_(—)3159 are catalytically active, with the smallerform providing a higher synergistic response, again corresponding to aspecific activity increase associated with size minimization.

Example 14 The Function of ActE Xylanases can be Assigned by FunctionalAssay of Proteins Produced by Using Cell-Free Translation

FIG. 29 shows that both of the xylanases identified in the fractions ofActE secretomes obtained from ion exchange chromatography can also beexpressed using cell-free translation and demonstrated to be xylanasesby catalytic activity assays. These proteins are SACTE_(—)0265 andSACTE_(—)0358. Other proteins that are not secreted were successfullyexpressed (SACTE_(—)2548, SACTE_(—)2286, SACTE_(—)437) as controlproteins, and as expected from their predicted intracellularlocalization, none of these controls exhibited xylanase activity. Thenegative result with the control proteins also demonstrates that thewheat germ extract used for cell-free translation of novel cellulolyticenzymes does not have an endogenous xylanase activity, as established inUS Patent Application Publication No.: US2010/037094 (Fox and Elsen).

Example 15 Total Protein Secreted by ActE can be Increased

A minimal set of enzymes for biomass deconstruction can be defined bycombining the additional enzymes whose expression is elicited duringgrowth on biomass (Table 1) with enzymes uniquely expressed duringgrowth on cellulose and xylan.

Besides assembling the proper enzymatic constituents, the level of totalprotein secreted is an important biotechnological constraint forindustrial enzyme production. FIG. 30 shows the non-optimized level ofsecreted protein obtained from growth of ActE on different biomasssubstrates. By use of lignocellulosic substrates for growth, secretedprotein levels up to 0.25 g per liter of culture medium can be readilyobtained. Growth on non-polymeric substrates such as cellobiose does notelicit a secreted protein response. FIG. 15, FIG. 16 and Table 1indicate that the twin-arginine pathway (Tat) is used during growth,thus identifying this pathway as playing a key role in the secretion ofenzymes required for extracellular deconstruction of biomasspolysaccharides (Natale et al., 2008; Chater et al., 2010). Methods toincrease the titer of secreted proteins are known, and have been highlyeffectively when applied to Streptomyces and other organisms (Cereghinoet al., 2002; Zhang et al., 2006; Nijland and Kuipers, 2008; Chater etal., 2010; Schuster and Schmoll, 2010). These established methods can beapplied to ActE to obtain more concentrated secretome preparations.

Example 16 ActE Enzymatic Activity Corresponds with Optimal GrowthConditions of Fermentation Organisms

FIG. 31 shows the temperature versus activity profile for ActEsecretomes for reaction with cellulose, xylan and mannan. These profilesare well matched to the growth optima range for mesophilic fermentationorganisms such as Saccharomyces cerevisiae, Zymomonas mobilis,Escherichia coli or others (Jarboe et al., 2010; Peralta-Yahya andKeasling, 2010), which are widely used for ethanol production from sugarhydrolysates. These hydrolysates are produced from biomass by theenzymatic action of highly cellulolytic secretomes, such as thosedescribed here from ActE. These optima are also well matched with theconditions found in the rumen, where the efficiency of conversion ofanimal feed, which is a biomass material, can be improved by addition ofenzymes.

FIG. 32 shows the pH versus activity profiles for ActE secretomes forreaction with cellulose, xylan and mannan. These profiles are wellmatched to the growth optima range for fermentation organisms such as S.cerevisiae, Z. mobilis, E. coli or other organisms (Jarboe et al., 2010;Peralta-Yahya and Keasling, 2010) which are widely used for ethanolproduction from sugar hydrolysates such as might be produced frombiomass by a highly cellulolytic secretome, such as those described herefrom ActE. These optima are also well matched with the conditions foundin the rumen, where the efficiency of conversion of animal feed, whichis a biomass material, can be improved by addition of enzymes. The ActEsecretome retains high specific activity (>80% of maximal) at pH 7,which closely approximates that of the rumen. Sectetomes from fungi suchas T. reesei are considerably less active at neutral pH, rendering themless effective at neutral pH.

The high cellulolytic capacity of ActE, and its correspondingsecretomes, coupled with the temperature and pH optima described abovepermit assembly of two-part systems to effect the simultaneousdeconstruction of biomass and fermentation to fuels.

Example 17 ActE Induction in Medium Containing Various Percentages ofCellulose

To determine ActE's growth profile on cellulose as a carbon source ActEwas grown in M63 media plus 5 g/L carbon. The carbon source ratio wasadjusted from 100% cellulose to 100% glucose, total carbon in eachculture was equal. Cells were grown for 6 days at 30 degrees.Supernatant was harvested, filtered, and separated by 4-20% SDS-PAGE.Results suggest that ActE is induced in media containing as little as20% cellulose, with optimal induction in medium containing between80%-100% cellulose (FIG. 33).

Example 18 Discussion

The work presented here provides the first genome-wide insight into howan aerobic microbe deconstructs polysaccharides. ActE achieves efficientutilization of cellulose by a simple combination of well-understoodhydrolytic reactions with newly identified oxidative reactions. The tworequired exoglucanases are each encoded by a single gene, which alsorepresents the only example of their respective GH families in thegenome. The proteins encoded by these genes provide reactions that arecomplementary to the reactions of other enzymes in the secretome, andprovide cellobiose as the major product of reaction. We have discoveredthat many of the highly abundant enzymes secreted by ActE during growthon cellulose have reduced size relative to their orthologs from closelyrelated organisms. This novel finding suggests natural evolution toimprove specific activity has already occurred in ActE in response togrowth in the highly specialized insect association. Additionalspecializations of ActE were identified by demonstrating the secretionof a unique set of proteins in response to biomass. In addition, thiswork defines how simple new combinations of improved biomassdeconstruction enzymes can be assembled according to the propensities ofthe naturally evolved system.

The present work also indicates that insect-associated microbes such asActE are important contributors to the vigorous attack on biomass byinsects. The ‘highly invasive’ designation given to Sirex has beengenerally attributed to the combined action of wasp and fungus (Tabataand Abe, 2000; Bergeron et al., 2011). Species convergence is nowrecognized in the microbial communities associated with insects (Suen etal., 2010; Hulcr et al., 2011). Given the ubiquitous presence ofStreptomycetes in these communities, the enzymatic properties describedhere also contribute a potential risk to pine forests, including thoseused for industrial purposes.

The invention has been described in connection with what are presentlyconsidered to be the most practical and preferred embodiments. However,the present invention has been presented by way of illustration and isnot intended to be limited to the disclosed embodiments. Accordingly,those skilled in the art will realize that the invention is intended toencompass all modifications and alternative arrangements within thespirit and scope of the invention as set forth in the appended claims.

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SEQUENCE LISTING

FIG. 19 is a pdf file containing the nucleic acid sequences of the ActEgenes disclosed herein.

FIG. 20 is a pdf file containing the amino acid sequences of the ActEgenes disclosed herein.

A separated txt file containing both the amino acid and nucleic acidsequences for all the claimed ActE genes in the claim is submitted alongwith the non-provisional application.

We claim:
 1. A method of digesting a lignocellulosic material, comprising the step of exposing the material to an effective amount of Streptomyces sp: ActE secretome preparation such that at least partial lignocellulosic digestion occurs.
 2. The method of claim 1 wherein the preparation is a supernatant preparation obtained from a Streptomyces sp. ActE culture.
 3. The method of claim 1, wherein the preparation is obtained from Streptomyces sp. ActE grown on a substrate wherein at least 40%, preferably 85%, of Streptomyces sp. ActE's carbon source in the substrate is derived from a material selected from the group consisting of cellulose, cellulose/hemicelluloses mixture, hemicelluloses, xylan, non-wood biomass, wood biomass, and chitin.
 4. The method of claim 1, wherein the lignocellulosic material is selected from the group consisting of materials that comprise at least 75% cellulose, cellulose/hemicelluloses, xylose, biomass and chitin.
 5. A purified preparation comprising the Streptomyces sp. ActE secretome.
 6. The preparation of claim 5 wherein the preparation is a supernatant preparation obtained from a Streptomyces sp. ActE culture.
 7. The preparation of claim 5 wherein Streptomyces sp. ActE is grown on a substrate wherein at least 40%, preferably 85%, of Streptomyces sp. ActE's carbon source in the substrate is derived from a material selected from the group consisting of cellulose, cellulose/hemicelluloses mixture, hemicelluloses, xylan, non-wood biomass, wood biomass, and chitin.
 8. A composition useful for digesting lignocellulosic material comprising SActE_(—)0237 (GH6) (SEQ ID NOs:1 and 17) gene or expression product thereof.
 9. The composition of claim 8 useful for biomass degradation, wherein the composition additionally comprises at least one member selected from the group consisting of SActE_(—)0357 (CE4) (SEQ ID NOs:7 and 23), SActE_(—)0358 (GH11) (SEQ ID NOs:8 and 24), SActE_(—)1310 (PL3) (SEQ ID NOs:9 and 25), SActE_(—)3717 (GH9) (SEQ ID NOs:10 and 26), SActE_(—)4638 (SEQ ID NOs:11 and 27), SActE_(—)4738 (GH16) (SEQ ID NOs:12 and 28), SActE_(—)4755 (GH64) (SEQ ID NOs:13 and 29), SActE_(—)5457 (GH46) (SEQ ID NOs:14 and 30), SActE_(—)5647 (GH87) (SEQ ID NOs:15 and 31), and SActE_(—)5978 (PL1) (SEQ ID NOs:16 and 32) genes or expression products derived thereof.
 10. A composition useful for digesting lignocellulosic material comprising SActE_(—)0236 (GH48) (SEQ ID NOs:2 and 18) gene or expression product thereof.
 11. The composition of claim 10 useful for biomass degradation, wherein the composition additionally comprises at least one member selected from the group consisting of SActE_(—)0357 (CE4) (SEQ ID NOs:7 and 23), SActE_(—)0358 (GH11) (SEQ ID NOs:8 and 24), SActE_(—)1310 (PL3) (SEQ ID NOs:9 and 25), SActE_(—)3717 (GH9) (SEQ ID NOs:10 and 26), SActE_(—)4638 (SEQ ID NOs:11 and 27), SActE_(—)4738 (GH16) (SEQ ID NOs:12 and 28), SActE_(—)4755 (GH64) (SEQ ID NOs:13 and 29), SActE_(—)5457 (GH46) (SEQ ID NOs:14 and 30), SActE_(—)5647 (GH87) (SEQ ID NOs:15 and 31), and SActE_(—)5978 (PL1) (SEQ ID NOs:16 and 32) genes or expression products derived thereof.
 12. A composition useful for digesting lignocellulosic material comprising SActE_(—)3159 (CBM33) (SEQ ID NOs:3 and 19) gene or expression product thereof.
 13. The composition of claim 12 useful for biomass degradation, wherein the composition additionally comprises at least one member selected from the group consisting of SActE_(—)0357 (CE4) (SEQ ID NOs:7 and 23), SActE_(—)0358 (GH11) (SEQ ID NOs:8 and 24), SActE_(—)1310 (PL3) (SEQ ID NOs:9 and 25), SActE_(—)3717 (GH9) (SEQ ID NOs:10 and 26), SActE_(—)4638 (SEQ ID NOs:11 and 27), SActE_(—)4738 (GH16) (SEQ ID NOs:12 and 28), SActE_(—)4755 (GH64) (SEQ ID NOs:13 and 29), SActE_(—)5457 (GH46) (SEQ ID NOs:14 and 30), SActE_(—)5647 (GH87) (SEQ ID NOs:15 and 31), and SActE_(—)5978 (PL1) (SEQ ID NOs:16 and 32) genes or expression products derived thereof.
 14. A composition useful for digesting lignocellulosic material comprising SActE_(—)0482 (GH5) (SEQ ID NOs:4 and 20) gene or expression product thereof.
 15. The composition of claim 14 useful for biomass degradation, wherein the composition additionally comprises at least one member selected from the group consisting of SActE_(—)0357 (CE4) (SEQ ID NOs:7 and 23), SActE_(—)0358 (GH11) (SEQ ID NOs:8 and 24), SActE_(—)1310 (PL3) (SEQ ID NOs:9 and 25), SActE_(—)3717 (GH9) (SEQ ID NOs:10 and 26), SActE_(—)4638 (SEQ ID NOs:11 and 27), SActE_(—)4738 (GH16) (SEQ ID NOs:12 and 28), SActE_(—)4755 (GH64) (SEQ ID NOs:13 and 29), SActE_(—)5457 (GH46) (SEQ ID NOs:14 and 30), SActE_(—)5647 (GH87) (SEQ ID NOs:15 and 31), and SActE_(—)5978 (PL1) (SEQ ID NOs:16 and 32) genes or expression products derived thereof.
 16. A method for digesting a lignocellulosic material, comprising exposing the material to a sufficient amount of a composition of claim 8, wherein the exposed material is at least partially digested.
 17. A method for digesting a lignocellulosic material, comprising exposing the material to a sufficient amount of a composition of claim 9, wherein the exposed material is at least partially digested.
 18. A method for digesting a lignocellulosic material, comprising exposing the material to a sufficient amount of a composition of claim 10, wherein the exposed material is at least partially digested.
 19. A method for digesting a lignocellulosic material, comprising exposing the material to a sufficient amount of a composition of claim 11, wherein the exposed material is at least partially digested.
 20. A method for digesting a lignocellulosic material, comprising exposing the material to a sufficient amount of a composition of claim 12, wherein the exposed material is at least partially digested.
 21. A method for digesting a lignocellulosic material, comprising exposing the material to a sufficient amount of a composition of claim 13, wherein the exposed material is at least partially digested.
 22. A method for digesting a lignocellulosic material, comprising exposing the material to a sufficient amount of a composition of claim 14, wherein the exposed material is at least partially digested.
 23. A method for digesting a lignocellulosic material, comprising exposing the material to a sufficient amount of a composition of claim 15, wherein the exposed material is at least partially digested.
 24. A purified preparation of Streptomyces sp. ActE, wherein the Streptomyces sp. ActE has been grown on a substrate wherein at least 40%, preferably 85%, of Streptomyces sp. ActE's carbon source in the substrate is derived from a material selected from the group consisting of cellulose, cellulose/hemicelluloses mixture, hemicelluloses, xylan, non-wood biomass, wood biomass and chitin.
 25. A purified preparation of Streptomyces sp. ActE, wherein the Streptomyces sp. ActE has been grown on a substrate wherein at least 40%, preferably 85%, of Streptomyces sp. ActE's carbon in the substrate is derived from pretreated lignocellulosic material.
 26. The preparation of claim 25, wherein the pretreated material has been exposed to pretreatment selected from the group consisting of acid hydrolysis, steam explosion, ammonia fiber expansion (AFEX), organosolve, sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL), ionic liquids (IL), metal-catalyzed hydrogen peroxide treatment, alkaline wet oxidation and ozone pretreatment. 